Plants Modified With Mini-Chromosomes

ABSTRACT

The invention is generally related to methods of generating plants transformed with novel autonomous mini-chromosomes. Mini-chromosomes with novel compositions and structures are used to transform plants cells which are in turn used to generate the plant. Methods for generating the plant include methods for delivering the mini-chromosome into plant cell to transform the cell, methods for selecting the transformed cell, and methods for isolating plants transformed with the mini-chromosome. Plants generated in the present invention contain novel genes introduced into their genome by integration into existing chromosomes.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/547,256 filed Feb. 23, 2004, which is incorporated by referenceherein in its entirety.

BACKGROUND OF THE INVENTION

Two general approaches are used for introduction of new geneticinformation (“transformation”) into cells. One approach is to introducethe new genetic information as part of another DNA molecule, referred toas an “episomal vector,” or “mini-chromosome”, which can be maintainedas an independent unit (an episome) apart from the host chromosomal DNAmolecule(s). Episomal vectors contain all the necessary DNA sequenceelements required for DNA replication and maintenance of the vectorwithin the cell. Many episomal vectors are available for use inbacterial cells (for example, see Maniatis et al., “Molecular Cloning: aLaboratory Manual, “Cold Spring Harbor Laboratory, Cold Sprint Harbor,N.Y. 1982.). However, only a few episomal vectors that function inhigher eukaryotic cells have been developed. Higher eukaryotic episomalvectors were primarily based on naturally occurring viruses. In higherplant systems gemini viruses are double-stranded DNA viruses thatreplicate through a double-stranded intermediate upon which an episomalvector could be based, although the gemini virus is limited to anapproximately 800 bp insert. Although an episomal plant vector based onthe Cauliflower Mosaic Virus has been developed, its capacity to carrynew genetic information also is limited (Brisson et al., Nature,310:511,1984).

The other general method of genetic transformation involves integrationof introduced DNA sequences into the recipient cell's chromosomes,permitting the new information to be replicated and partitioned to thecell's progeny as a part of the natural chromosomes. The introduced DNAusually is broken and joined together in various combinations before itis integrated at random sites into the cell's chromosome (see, forexample Wigler et al., Cell, 11:223, 1977). Common problems with thisprocedure are the rearrangement of introduced DNA sequences andunpredictable levels of expression due to the location of the transgenein the genome or so called “position effect variegation” (Shingo et al.,Mol. Cell. Biol., 6:1787, 1986). Further, unlike episomal DNA,integrated DNA cannot normally be precisely removed. A more refined formof integrative transformation can be achieved by exploiting naturallyoccurring viruses that integrate into the host's chromosomes as part oftheir life cycle, such as retroviruses (see Chepko et al., Cell,37:1053, 1984).

One common genetic transformation method used in higher plants is basedon the transfer of bacterial DNA into plant chromosomes that occursduring infection by the phytopathogenic soil bacterium Agrobacterium(see Nester et al, Ann. Rev. Plant Phys., 35:387-413, 1984). Bysubstituting genes of interest for the naturally transferred bacterialsequences (called T-DNA), investigators have been able to introduce newDNA into plant cells. However, even this more “refined” integrativetransformation system is limited in three major ways. First, DNAsequences introduced into plant cells using the Agrobacterium T-DNAsystem are frequently rearranged (see Jones et al., Mol Gen. Genet.,207:478, 1987). Second, the expression of the introduced DNA sequencesvaries between individual transformants (see Jones et al, EMBO J.,4:2411-2418, 1985). This variability is presumably caused by rearrangedsequences and the influence of surrounding sequences in the plantchromosome (i.e., position effects), as well as methylation of thetransgene. Finally, insertion of extra elements into the genome candisrupt the genes, promoters or other genetic elements necessary fornormal plant growth and function.

Another widely used technique to genetically transform plants involvesthe use of micro-projectile bombardment. In this process, a nucleic acidcontaining the desired genetic elements to be introduced into the plantis deposited on or in small metallic particles, e.g., tungsten,platinum, or preferably gold, which are then delivered at a highvelocity into the plant tissue or plant cells. However, similar problemsarise as with Agrobacterium-mediated gene transfer, and as noted aboveexpression of the inserted DNA can be unpredictable and insertion ofextra elements into the genome can disrupt and adversely impact plantprocesses.

One attractive alternative to commonly used methods of transformation isthe use of an artificial chromosome. Artificial chromosomes are man-madelinear or circular DNA molecules constructed in part from cis-acting DNAsequence elements that provide replication and partitioning of theconstructed chromosomes (see Murray et al., Nature, 305:189-193, 1983).Desired elements include: (1) origin of replication, which are the sitesfor initiation of DNA replication, (2) centromeres (site of kinetochoreassembly and responsible for proper distribution of replicatedchromosomes into daughter cells at mitosis or meiosis), and (3) if thechromosome is linear, telomeres (specialized DNA structures at the endsof linear chromosomes that function to stabilize the ends and facilitatethe complete replication of the extreme termini of the DNA molecule). Anadditional desired element is a chromatin organizing sequence. It iswell documented that centromere function is crucial for stablechromosomal inheritance in almost all eukaryotic organisms (reviewed inNicklas 1988). The centromere accomplishes this by attaching, viacentromere binding proteins, to the spindle fibers during mitosis andmeiosis, thus ensuring proper gene segregation during cell divisions.

The essential chromosomal elements for construction of artificialchromosomes have been precisely characterized in lower eukaryoticspecies, and more recently in mouse and human. Autonomous replicationsequences (ARSs) have been isolated from unicellular fungi, includingSaccharomyces cerevisiae (brewer's yeast) and Schizosaccharomyces pombe(see Stinchcomb et al, 1979 and Hsiao et al, 1979). An ARS behaves likea origin of replication allowing DNA molecules that contain the ARS tobe replicated in concert with the rest of the genome after introductioninto the cell nuclei of these fungi. DNA molecules containing thesesequences replicate, but in the absence of a centromere they are notpartitioned into daughter cells in a controlled fashion that ensuresefficient chromosome inheritance.

Artificial chromosomes have been constructed in yeast using the threecloned essential chromosomal elements (see Murray et al., Nature,305:189-193, 1983). None of the essential components identified inunicellular organisms, however, function in higher eukaryotic systems.For example, a yeast centromere sequence will not confer stableinheritance upon vectors transformed into higher eukaryotes.

In contrast to the detailed studies done in yeast, less is known aboutthe molecular structure of functional centromeric DNA of highereukaryotes. Ultrastructural studies indicate that higher eukaryotickinetochores, which are specialized complexes of proteins that form onthe centromere during late prophase, are large structures (mammaliankinetochore plates are approximately 0.3 μm in diameter) which possessmultiple microtubule attachment sites (reviewed in Rieder, 1982). It istherefore possible that the centromeric DNA regions of these organismswill be correspondingly large, although the minimal amount of DNAnecessary for centromere function may be much smaller.

While the above studies have been useful in elucidating the structureand function of centromeres, it was not known whether informationderived from lower eukaryotic or mammalian higher eukaryotic organismswould be applicable to plants. There exists a need for clonedcentromeres from higher eukaryotic organisms, particularly plantorganisms, which would represent a first step in production ofartificial chromosomes. There further exists a need for plant cells,plants, seeds and progeny containing functional, stable, and autonomousartificial chromosomes capable of carrying a large number of differentgenes and genetic elements.

SUMMARY OF THE INVENTION

The invention provides for adchromosomal plants, described in furtherdetail herein, comprising a mini-chromosome, wherein saidmini-chromosome preferably has a transmission efficiency during mitoticdivision of at least 90%, for example, at least 95%. Additionally, theseadchromosomal plants may comprise a mini-chromosome having atransmission efficiency during meiotic division of, e.g., at least 80%,at least 85%, at least 90% or at least 95%.

In one embodiment, the adchromosomal plants of the invention comprise amini-chromosome that is 1000 kilobases or less in length. In exemplaryembodiments, the adchromosomal plant comprises a mini-chromosome that is600 kilobases or less in length or 500 kilobases or less in length.

In another embodiment, the mini-chromosome of any of the precedingadchromosomal plants of the invention comprises a site for site-specificrecombination.

In an embodiment, the mini-chromosome of any of the precedingadchromosomal plants of the invention comprises a centromeric nucleicacid insert derived from a crop plant centromere. In an exemplaryembodiment, the centromeric nucleic acid insert is derived from genomicDNA of a plant selected from the group consisting of Brassica,Nicotiana, Lycopersicum, Glycine or Zea species. In another exemplaryembodiment, the centromeric nucleic acid insert is derived from genomicDNA of a plant selected from the group consisting of broccoli, canola,tobacco, tomato, soybean or corn.

In another embodiment, the invention provides for the mini-chromosome ofany one of the preceding adchromosomal plants, further comprising acentromeric nucleic acid insert that comprises artificially synthesizedrepeated nucleotide sequences. These artificially synthesized repeatednucleotide sequences may be derived from natural centromere sequences,combinations or fragments of natural centromere sequences including acombination of repeats of different lengths, a combination of differentsequences, a combination of both different repeat lengths and differentsequences, a combination of repeats from two or more plant species, acombination of different artificially synthesized sequences or acombination of natural centromere sequence(s) and artificiallysynthesized sequence(s).

The invention also provides for a mini-chromosome of any of thepreceding adchromosomal plants of the invention, wherein themini-chromosome is derived from a donor clone or a centromere clone andhas substitutions, deletions, insertions, duplications or arrangementsof one or more nucleotides in the mini-chromosome compared to thenucleotide sequence of the donor clone or centromere clone. In oneembodiment, the mini-chromosome is obtained by passage of themini-chromosome through one or more hosts. In another embodiment, themini-chromosome is obtained by passage of the mini-chromosome throughtwo or more different hosts. The host may be selected from the groupconsisting of viruses, bacteria, yeasts, plants, prokaryotic organisms,or eukaryotic organisms.

The invention also provides for a mini-chromosome of any of thepreceding adchromosomal plants of the invention, wherein themini-chromosome comprises one or more exogenous nucleic acids. Infurther exemplary embodiments, the mini-chromosome comprises at leasttwo or more, at least three or more, at least four or more, at leastfive or more or at least ten or more exogenous nucleic acids.

In one embodiment, at least one exogenous nucleic acid of any of thepreceding mini-chromosomes of a plant is operably linked to aheterologous regulatory sequence functional in plant cells. Theinvention provides for exogenous nucleic acids linked to a plantregulatory sequence. The invention also provides for exogenous nucleicacids linked to a non-plant regulatory sequence, such as an inset oryeast regulatory sequence. Exemplary regulatory sequences comprise anyone of SEQ ID NOS: 4 to 23 or a functional fragment or variant thereof.

In another embodiment, the mini-chromosome of any of the precedingadchromosomal plants comprises an exogenous nucleic acid that confersherbicide resistance, insect resistance, disease resistance, or stressresistance on the plant. The invention provides for mini-chromosomescomprising an exogenous nucleic acid that confers resistance tophosphinothricin or glyphosate herbicide. The invention also providesfor mini-chromosomes comprising an exogenous nucleic acid that encodes aphosphinothricin acetyltransferase, glyphosate acetyltransferase or amutant enoylpyruvylshikimate phosphate (EPSP) synthase.

The invention also provides for the mini-chromosome of any of thepreceding adchromosomal plants comprising an exogenous nucleic acid thatencodes a Bacillus thuringiensis crystal toxin gene or Bacillus cereustoxin gene. The invention further provides for the mini-chromosome ofany of the preceding adchromosomal plants comprising an exogenousnucleic acid that confers resistance to drought, heat, chilling,freezing, excessive moisture, ultraviolet light, ionizing radiation,toxins, pollution, mechanical stress or salt stress. The invention alsoprovides for a mini-chromosome of any of the preceding adchromosomalplants that comprises an exogenous nucleic acid that confers resistanceto a virus, bacteria, fungi or nematode.

In another embodiment, the mini-chromosome of any of the precedingadchromosomal plants comprises an exogenous nucleic acid conferringherbicide resistance, an exogenous nucleic acid conferring insectresistance, and at least one additional exogenous nucleic acid.

The invention provides for mini-chromosomes of any of the precedingadchromosomal plants comprising an exogenous nucleic acid selected fromthe group consisting of a nitrogen fixation gene, a plant stress-inducedgene, a nutrient utilization gene, a gene that affects plantpigmentation, a gene that encodes an antisense or ribozyme molecule, agene encoding a secretable antigen, a toxin gene, a receptor gene, aligand gene, a seed storage gene, a hormone gene, an enzyme gene, aninterleukin gene, a clotting factor gene, a cytokine gene, an antibodygene, a growth factor gene, a transcription factor gene, atranscriptional repressor gene, a DNA-binding protein gene, arecombination gene, a DNA replication gene, a programmed cell deathgene, a kinase gene, a phosphatase gene, a G protein gene, a cyclingene, a cell cycle control gene, a gene involved in transcription, agene involved in translation, a gene involved in RNA processing, a geneinvolved in RNAi, an organellar gene, a intracellular trafficking gene,an integral membrane protein gene, a transporter gene, a membranechannel protein gene, a cell wall gene, a gene involved in proteinprocessing, a gene involved in protein modification, a gene involved inprotein degradation, a gene involved in metabolism, a gene involved inbiosynthesis, a gene involved in assimilation of nitrogen or otherelements or nutrients, a gene involved in controlling carbon flux, geneinvolved in respiration, a gene involved in photosynthesis, a geneinvolved in light sensing, a gene involved in organogenesis, a geneinvolved in embryogenesis, a gene involved in differentiation, a geneinvolved in meiotic drive, a gene involved in self incompatibility, agene involved in development, a gene involved in nutrient, metabolite ormineral transport, a gene involved in nutrient, metabolite or mineralstorage, a calcium-binding protein gene, or a lipid-binding proteingene.

The invention also provides for a mini-chromosome of any of thepreceding adchromosomal plants comprising an exogenous enzyme geneselected from the group consisting of a gene that encodes an enzymeinvolved in metabolizing biochemical wastes for use in bioremediation, agene that encodes an enzyme for modifying pathways that producesecondary plant metabolites, a gene that encodes an enzyme that producesa pharmaceutical, a gene that encodes an enzyme that improves changesthe nutritional content of a plant, a gene that encodes an enzymeinvolved in vitamin synthesis, a gene that encodes an enzyme involved incarbohydrate, polysaccharide or starch synthesis, a gene that encodes anenzyme involved in mineral accumulation or availability, a gene thatencodes a phytase, a gene that encodes an enzyme involved in fatty acid,fat or oil synthesis, a gene that encodes an enzyme involved insynthesis of chemicals or plastics, a gene that encodes an enzymeinvolved in synthesis of a fuel and a gene that encodes an enzymeinvolved in synthesis of a fragrance, a gene that encodes an enzymeinvolved in synthesis of a flavor, a gene that encodes an enzymeinvolved in synthesis of a pigment or dye, a gene that encodes an enzymeinvolved in synthesis of a hydrocarbon, a gene that encodes an enzymeinvolved in synthesis of a structural or fibrous compound, a gene thatencodes an enzyme involved in synthesis of a food additive, a gene thatencodes an enzyme involved in synthesis of a chemical insecticide, agene that encodes an enzyme involved in synthesis of an insectrepellent, or a gene controlling carbon flux in a plant.

In an embodiment of the invention, the mini-chromosomes of any one ofthe preceding adchromosomal plants comprise n copies of a repeatednucleotide sequence, wherein n is less than 1000. In other exemplaryembodiments, the mini-chromosomes of the plants comprise n copies of arepeated nucleotide sequence, wherein n is at least 5, wherein n is atleast 15, or wherein n is at least 50.

In another embodiment of the invention, the mini-chromosomes of any ofthe preceding adchromosomal plants comprise a telomere.

The invention also provides embodiments wherein the mini-chromosome ofany of the preceding adchromosomal plants is circular.

In one embodiment of the invention, any of the preceding adchromosomalplants are a monocotyledon. In another embodiment of the invention, anyof the preceding adchromosomal plants are a dicotyledone. The inventionalso provides that the adchromosomal plants of the invention are, e.g.,crop plants, cereal plants, vegetable crops, field crops, fruit and vinecrops, wood or fiber crops or ornamental plants. The invention alsoprovides exemplary adchromosomal plants that are Brassica, Nicotiana,Lycopersicum, Glycine or Zea species.

Another embodiment of the invention is a part of any of the precedingadchromosomal plants. Exemplary plant parts of the invention include apod, root, cutting, tuber, stem, stalk, fruit, berry, nut, flower, leaf,bark, wood, epidermis, vascular tissue, organ, protoplast, crown, callusculture, petiole, petal, sepal, stamen, stigma, style, bud, meristem,cambium, cortex, pith, sheath, silk or embryo. Other exemplary plantparts are a meiocyte or gamete or ovule or pollen or endosperm of any ofthe preceding adchromosomal plants. Other exemplary plant parts are aseed, embryo or propagule of any of the preceding adchromosomal plants.

An embodiment of the invention is a progeny of any of the precedingadchromosomal plants of the invention. These progeny of the inventionmay be the result of self-breeding, cross-breeding, apomyxis or clonalpropagation. In exemplary embodiments, the invention also provides forprogeny that comprise a mini-chromosome that is descended from aparental mini-chromosome that contained a centromere less than 150kilobases in length, less than 100 kilobases in length or less than 50kilobases in length.

In another aspect, the invention provides for methods of making amini-chromosome for use in any of the preceding adchromosomal plants ofthe invention. These methods comprise identifying a centromerenucleotide sequence in a genomic DNA library using a multiplicity ofdiverse probes, and constructing a mini-chromosome comprising thecentromere nucleotide sequence. These methods may further comprisedetermining hybridization scores for hybridization of the multiplicityof diverse probes to genomic clones within the genomic nucleic acidlibrary, determining a classification for genomic clones within thegenomic nucleic acid library according to the hybridization scores forat least two of the diverse probes, and selecting one or more genomicclones within one or more classifications for constructing themini-chromosome.

In exemplary embodiments, the step of determining a classification forgenomic clones within the genomic nucleic acid library may utilize thehybridization scores for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 ormore different probes. A classification may comprise a pattern of high,medium or low hybridization scores to various probes.

Exemplary embodiments of probes useful in this method include a probethat hybridizes to the centromere region of a chromosome, a probe thathybridizes to satellite repeat DNA, a probe that hybridizes toretroelement DNA, a probe that hybridizes to portions of genomic DNAthat are heavily methylated, a probe that hybridizes to arrays of tandemrepeats in genomic DNA, a probe that. hybridizes to telomere DNA or aprobe that hybridizes to a pseudogene. Other exemplary probes include, aprobe that hybridizes to ribosomal DNA, a probe that hybridizes tomitochondrial DNA, or a probe that hybridizes to chloroplast DNA, forwhich preferably a classification comprises a low hybridization scorefor hybridization to said probe.

Another aspect of the invention provides for methods of making any oneof the preceding adchromosomal plants comprising delivering amini-chromosome to a plant cell using a biolistic method, wherein aparticle suitable for use in biolistic method is delivered in a liquidwith the mini-chromosome, and regenerating a plant from the plant cell.The liquid may further comprise a divalent ion and a di- or poly-amine.In exemplary embodiments, the liquid comprises water, CaCl₂, andspermidine, and the particles are gold particles. Suitable alternativesto spermidine are, e.g., spermine or other aliphatic or conjugated di-or poly-amines such as 1,5-diaminopentane, 1,6-diaminohexane,1,7-diaminoheptane, 1,8-diaminooctane, histamine or related molecules.

A further aspect of the invention provides for methods of making any ofthe preceding adchromosomal plant comprising co-delivering to a plantcell a mini-chromosome and a nucleic acid encoding a growth inducinggene, wherein said nucleic acid is not part of the mini-chromosome, andregenerating a plant from the plant cell. The invention further providesfor methods comprising co-delivering a nucleic acid encoding a growthinducing gene is not expressed or alternatively is not present in theregenerated plant. The invention also provides for methods wherein theco-delivered nucleic acid encodes a growth inducing gene expressedduring regeneration. The growth inducing gene may a plant growthregulator gene, an organogenesis-promoting gene, anembryogenesis-promoting gene or regeneration-promoting gene, such asAgrobacterium tumefaciens isopentenyl transferase gene, Agrobacteriumrhizogenes isopentenyl transferase gene, Agrobacterium tumefaciensindole-3-acetamide hydrolase (IAAH) gene or Agrobacterium tumefacienstyptophan-2-monooxygenase (IAAM) gene.

Another aspect of the invention provides for methods of using any of thepreceding adchromosomal plants for a food product, a pharmaceuticalproduct or chemical product, according to which a suitable exogenousnucleic acid is expressed in adchromosomal plants or plant cells and theplant or plant cells are grown. The plant may secrete the product intoits growth environment or the product may be contained within the plant,in which case the plant is harvested and desirable products areextracted.

Thus, the invention contemplates methods of using any of the precedingadchromosomal plants to produce a modified food product, for example, bygrowing a plant that expresses a exogenous nucleic acid that alters thenutritional content of the plant, and harvesting or processing the cornplant.

The invention also contemplates methods of using any of the precedingadchromosomal plants to produce a recombinant protein, by growing aplant comprising a mini-chromosome that comprises an exogenous nucleicacid encoding the recombinant protein. Optionally the plant is harvestedand the desired recombinant protein is isolated from the plant.Exemplary recombinant proteins include pharmaceutical proteins orindustrial enzymes.

The invention also contemplates methods of using any of the precedingadchromosomal plants to produce a recombinant protein, by growing aplant comprising a mini-chromosome that comprises an exogenous nucleicacid encoding an enzyme involved in synthesis of the chemical product.Optionally the plant is harvested and the desired chemical product isisolated from the plant. Exemplary chemical products includepharmaceutical products.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an example of a mini-chromosome vector in the presentinvention containing 2 genes;

FIG. 2 is another example of a mini-chromosome vector in the presentinvention containing 4 genes;

FIG. 3 is a mini-chromosome from which all bacterial sequences have beenremoved. In this embodiment, bacterial sequence present between or amongthe plant-expressed genes or other mini-chromosome sequences would beexcised prior to removal of the remaining bacterial sequences, bycutting the mini-chromosome with endonuclease #1, and re-ligating thestructure such that the antibiotic-resistance gene #1 has been lost.

FIG. 4 shows various structural configurations by which mini-chromosomeelements can be oriented with respect to each other.

FIG. 5 shows the alignment of Brassica consensus centromere satelliterepeats.

FIG. 6 shows the alignment of Glycine max (soybean) consensus centromeresatellite repeats.

DETAILED DESCRIPTION OF THE INVENTION

While this invention is susceptible of embodiment in many differentforms, there is shown in the drawings, and will be described herein indetail, specific embodiments thereof with the understanding that thepresent disclosure is to be considered as an exemplification of theprinciples of the invention and is not intended to limit the inventionto the specific embodiments illustrated.

The invention is based on the production of modified plants, containingfunctional, stable, autonomous mini-chromosomes. Such mini-chromosomeshave been shown herein to be meiotically transmitted to progeny.

One aspect of the invention is related to plants containing functional,stable, autonomous mini-chromosomes, preferably carrying one or morenucleic acids exogenous to the cell. Such plants carryingmini-chromosomes are contrasted to transgenic plants whose genome hasbeen altered by chromosomal integration of an exogenous nucleic acid.Preferably, expression of the exogenous nucleic acid, eitherconstitutively or in response to a signal which may be a challenge or astimulus, results in an altered phenotype of the plant.

The invention provides for mini-chromosomes comprising at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 250, 500, 1000 or more exogenous nucleic acids.

The invention contemplates that any plants, including but not limited tomonocots, dicots, gymnosperm, field crops, vegetable crops, fruit andvine crops, or any specific plants named herein, may be modified bycarrying autonomous mini-chromosomes as described herein. A relatedaspect of the invention is plant parts or plant tissues, includingpollen, silk, endosperm, ovule, seed, embryo, pods, roots, cuttings,tubers, stems, stalks, fruit, berries, nuts, flowers, leaves, bark,whole plant, plant cell, plant organ, protoplast, cell culture, or anygroup of plant cells organized into a structural and functional unit,any cells of which carry mini-chromosomes.

A related aspect of the invention is adchromosomal plant parts or planttissues, including pollen, silk, endosperm, ovule, seed, embryo, pods,roots, cuttings, tubers, stems, stalks, crown, callus culture, petiole,petal, sepal, stamen, stigma, style, bud, fruit, berries, nuts, flowers,leaves, bark, wood, whole plant, plant cell, plant organ, protoplast,cell culture, or any group of plant cells organized into a structuraland functional unit. In one preferred embodiment, the exogenous nucleicacid is primarily expressed in a specific location or tissue of a plant,for example, epidermis, vascular tissue, meristem, cambium, cortex,pith, leaf, sheath, flower, root or seed. Tissue-specific expression canbe accomplished with, for example, localized presence of themini-chromosome, selective maintenance of the mini-chromosome, or withpromoters that drive tissue-specific expression.

Another related aspect of the invention is meiocytes, pollen, ovules,endosperm, seed, somatic embryos, apomyctic embryos, embryos derivedfrom fertilization, vegetative propagules and progeny of the originallyadchromosomal plant and of its filial generations that retain thefunctional, stable, autonomous mini-chromosome. Such progeny includeclonally propagated plants, embryos and plant parts as well as filialprogeny from self- and cross-breeding, and from apomyxis.

Preferably the mini-chromosome is transmitted to subsequent generationsof viable daughter cells during mitotic cell division with atransmission efficiency of at least 60%, 70%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99%. More preferably, the mini-chromosome is transmitted toviable gametes during meiotic cell division with a transmissionefficiency of at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% when more than one copy of the mini-chromosome is present in thegamete mother cells of the plant. Preferably, the mini-chromosome istransmitted to viable gametes during meiotic cell division with atransmission frequency of at least 20%, 30%, 40%, 45%, 46%, 47%, 48%, or49% when one copy of the mini-chromosome is present in the gamete mothercells of the plant. For production of seeds via sexual reproduction orby apomyxis the mini-chromosome is preferably transferred into at least60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of viable embryoswhen cells of the plant contain more than one copy of themini-chromosome. For production of seeds via sexual reproduction or byapomyxis from plants with one mini-chromosome per cell, themini-chromosome is preferably transferred into at least 20%, 30%, 40%,45%, 46%, 47%, 48%, or 49% of viable embryos.

Preferably, a mini-chromosome that comprises an exogenous selectabletrait or exogenous selectable marker can be employed to increase thefrequency in subsequent generations of adchromosomal cells, tissues,gametes, embryos, endosperm, seeds, plants or progeny. More preferably,the frequency of transmission of mini-chromosomes into viable cells,tissues, gametes, embryos, endospern, seeds, plants or progeny can be atleast 95%, 96%, 97%, 98%, 99% or 99.5% after mitosis or meiosis byapplying a selection that favors the survival of adchromosomal cells,tissues, gametes, embryos, endosperm, seeds, plants or progeny over suchcells, tissues, gametes, embryos, endosperm, seeds, plants or progenylacking the mini-chromosome.

Transmission efficiency may be measured as the percentage of progenycells or plants that carry the mini-chromosome as measured by one ofseveral assays taught herein including detection of reporter genefluorescence, PCR detection of a sequence that is carried by themini-chromosome, RT-PCR detection of a gene transcript for a genecarried on the mini-chromosome, Western analysis of a protein producedby a gene carried on the mini-chromosome, Southern analysis of the DNA(either in total or a portion thereof) carried by the mini-chromosome,fluorescence in situ hybridization (FISH) or in situ localization byrepressor binding, to name a few. Any assay used to detect the presenceof the mini-chromosome (or a portion of the mini-chromosome) may be usedto measure the efficiency of a parental cell or plant transmits themini-chromosome to its progeny. Efficient transmission as measured bysome benchmark percentage should indicate the degree to which themini-chromosome is stable through the mitotic and meiotic cycles.

Plants of the invention may also contain chromosomally integratedexogenous nucleic acid in addition to the autonomous mini-chromosomes.The adchromosomal plants or plant parts, including plant tissues of theinvention may include plants that have chromosomal integration of someportion of the mini-chromosome in some or all cells the plant. Theplant, including plant tissue or plant cell is still characterized asadchromosomal despite the occurrence of some chromosomal integration. Inone aspect of the invention, the autonomous mini-chromosome can beisolated from integrated exogenous nucleic acid by crossing theadchromosomal plant containing the integrated exogenous nucleic acidwith plants producing some gametes lacling the integrated exogenousnucleic acid and subsequently isolating offspring of the cross, orsubsequent crosses, that are adchromosomal but lack the integratedexogenous nucleic acid. This independent segregation of themini-chromosome is one measure of the autonomous nature of themini-chromosome.

Another aspect of the invention relates to methods for producing andisolating such adchromosomal plants containing functional, stable,autonomous mini-chromosomes.

In one embodiment, the invention contemplates improved methods forisolating native centromere sequences. In another embodiment, theinvention contemplates methods for generating variants of native orartificial centromere sequences by passage through bacterial or plant orother host cells.

In a further embodiment, the invention contemplates methods fordelivering the mini-chromosome into plant cells or tissues to transformthe cells or tissues.

In yet another embodiment, the invention contemplates improved methodsfor regenerating plants, including methods for co-delivery of growthinducing genes with mini-chromosomes. The growth delivery genes includeAgrobacterium tumefaciens or A rhizogenes isopentenyl transferase (IPT)genes involved in cytokinin biosynthesis, plant isopentenyl transferase(IPT) genes involved in cytokinin biosynthesis (from any plant),Agrobacterium tumefaciens IAAH, IAAM genes involved in auxinbiosynthesis (indole-3-acetamide hydrolase andtryptophan-2-monooxygenase, respectively), Agrobacterium rhizogenesrolA, rolB and rolC genes involved in root formation, Agrobacteriumtumefaciens Aux1, Aux2 genes involved in auxin biosynthesis(indole-3-acetamide hydrolase or tryptophan-2-monooxygenase genes),Arabidopsis thaliana leafy cotyledon genes (e.g. Lec1, Lec2) promotingembryogenesis and shoot formation (see Stone et al., Proc. Natl Acad.Sci USA 98:

11806-11811), Arabidopsis thaliana ESR1 gene involved in shoot formation(see Banno et al., Plant Cell 13: 2609-2618), Arabidopsis thalianaPGA6/WUSCHEL gene involved in embryogenesis (see Zuo et al., Plant J.30: 349-359).

In yet a further embodiment, the invention contemplates methods forselecting modified plant cells or plant parts containingmini-chromosomes for regeneration. Such methods include assays foridentifying adchromosomal plants or cells by determining thatmini-chromosomes within the modified plant cell or plant are functional,stable, and autonomous. Exemplary assays for assessing mini-chromosomeperformance include lineage-based inheritance assays, use of chromosomeloss agents to demonstrate autonomy, global mitotic mini-chromosomeinheritance assays (sectoring assays) with or without the use of agentsinducing chromosomal loss, assays measuring expression levels of markergenes in the mini-chromosome over time and space in a plant, physicalassays for separation of autonomous mini-chromosomes from endogenousnuclear chromosomes of plants, molecular assays demonstrating conservedmini-chromosome structure, such as PCR, Southern blots, mini-chromosomerescue, cloning and characterization of mini-chromosome sequencespresent in the plant, cytological assays detecting mini-chromosomepresence in the cell's genome (e.g. FISH) and meiotic mini-chromosomeinheritance assays, which measure the levels of mini-chromosomeinheritance into a subsequent generation of plants via meiosis andgametes, embryos, endosperm or seeds.

The invention also contemplates novel methods of screening foradchromosomal plant cells that involve use of relatively low,sub-killing concentrations of selection agent (e.g. sub-killingantibiotic concentrations), and also involve use of a screenable marker(e.g., a visible marker gene) to identify clusters of modified cellscarrying the screenable marker, after which these screenable cells aremanipulated to homogeneity. Another aspect of the present invention isrelated to methods of making and compositions of non-plant promoters forexpressing genes in plants.

The invention further provides isolated promoter nucleic acid sequencescomprising any one of SEQ ID NOS: 4 to 23, or fragments or variantsthereof that retain expression-promoting activity. Mini-chromosomescomprising non-plant promoter sequences such as these that are operablylinked to plant-expressed genes (e.g., genes that confer a differentphenotype on plants), are contemplated as are plants comprising suchmini-chromosomes.

Another aspect is related to methods for using exonuclease to enrich forcircular mini-chromosome DNA in genomic DNA preparations.

Another aspect of the invention relates to methods for using suchadchromosomal plants containing a mini-chromosome for producing foodproducts, pharmaceutical products and chemical products by appropriateexpression of exogenous nucleic acid(s) contained within themini-chromosome(s).

It has also been shown herein that mini-chromosomes containingcentromeres from one plant species, when inserted into plant cells of adifferent species or even a different genus or family, can be stable,functional and autonomous. For example, as shown herein, a broccolicentromere (B. oleraceae) is functional in a canola (B. napus) plant.Similarly, a tomato (Lycopersicum) centromere is functional in a tobacco(Nicotiana) plant. A soybean (G. max) centromere is functional in abroccoli (B. oleraceae) and tobacco plant. Tobacco and tomato are in thesame family of Solanaceae plants. Soybean is in the Leguminoseae familyand broccoli is in the Brassicaceae family. Thus, another aspect of theinvention is an adchromosomal plant comprising a functional, stable,autonomous mini-chromosome that contains centromere sequence derivedfrom a different taxonomic plant species, or derived from a differenttaxonomic plant species, genus, family, order or class.

Yet another aspect of the invention provides novel autonomousmini-chromosomes with novel compositions and structures which are usedto transform plant cells which are in turn used to generate a plant (ormultiple plants). Exemplary mini-chromosomes of the invention arecontemplated to be of a size 2000 kb or less in length. Other exemplarysizes of mini-chromosomes include less than or equal to, e.g., 1500 kb,1000 kb, 900 kb, 800 kb, 700 kb, 600 kb, 500 kb, 450 kb, 400 kb, 350 kb,300 kb, 250 kb, 200 kb, 150 kb, 100 kb, 80 kb, 60 kb, or 40 kb inlength.

In a related aspect, novel centromere compositions as characterized bysequence content, size or other parameters are provided. Preferably, theminimal size of centromeric sequence is utilized in mini-chromosomeconstruction. Exemplary sizes include a centromeric nucleic acid insertderived from a portion of plant genomic DNA, that is less than or equalto 1000 kb, 900 kb, 800 kb, 700 kb, 600 kb, 500 kb, 400 kb, 300 kb, 200kb, 150 kb, 100 kb, 95 kb, 90 kb, 85 kb, 80 kb, 75 kb, 70 kb, 65 kb, 60kb, 55 kb, 50 kb, 45 kb, 40 kb, 35 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10kb, 5 kb, or 2 kb in length. For example, rescued functional variantsoybean centromeric sequences have been shown to be less than 30 kb insize. Another related aspect is the novel structure of themini-chromosome, particularly structures lacking bacterial sequences,e.g, required for bacterial propagation.

In exemplary embodiments the invention contemplates mini-chromosomes orother vectors comprising a repeated nucleotide sequence derived from aBrassica plant and adchromosomal plants or parts containing thesemini-chromosomes. Exemplary repeated nucleotide sequences include (1)SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 51 or SEQ ID NO: 52,or fragments or variants thereof, (2) combinations of any of theseBrassica sequences or a fragment or variant thereof with anotherBrassica-derived centromeric nucleotide sequence, (3) combinations ofany of these Brassica sequences or a fragment or variant thereof with acentromeric nucleotide sequence derived from a different plant species,and (4) combinations of any of the above with artificially synthesizedcentromeric nucleotide sequences.

In exemplary embodiments the invention also contemplatesmini-chromosomes or other vectors comprising a repeated nucleotidesequence derived from a Glycine max plant and adchromosomal plants orparts containing these mini-chromosomes. Exemplary repeated nucleotidesequences include (1) SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:26, orfragments or variants thereof, (2) combinations of any of these soybeansequences or a fragment or variant thereof with another soybean-derivedcentromeric nucleotide sequence, (3) combinations of any of thesesoybean sequences or a fragment or variant thereof with a centromericnucleotide sequence derived from a different plant species, and (4)combinations of any of the above with artificially synthesizedcentromeric nucleotide sequences.

In exemplary embodiments, the invention also contemplatesmini-chromosomes or other vectors comprising fragments or variants ofthe genomic DNA inserts of the BAC clones [identified as BB5, SB6, TB99,ZB19, or ZB113deposited on Feb. 23, 2005 with the American Type CultureCollection (ATCC), P.O. Box 1549 Manassas, Va. 20108, USA, underAccession Nos. ______, ______, ______, ______ and, ______,respectively], or naturally occurring descendants thereof, that retainthe ability to segregate during mitotic or meiotic division as describedherein, as well as adchromosomal plants or parts containing thesemini-chromosomes. Other exemplary embodiments include fragments orvariants of the genomic DNA inserts of any of the BAC clones identifiedherein, or descendants thereof, and fragments or variants of thecentromeric nucleic acid inserts of any of the vectors ormini-chromosomes identified herein.

In other exemplary embodiments, the invention contemplatesmini-chromosomes or other vectors comprising centromeric nucleotidesequence that when hybridized to 1, 2, 3, 4, 5, 6, 7, 8 or more of theprobes described in the examples herein, under hybridization conditionsdescribed herein, e.g. low, medium or high stringency, provides relativehybridization scores as described in the examples herein. Preferably theprobes for which relative hybridization scores are described herein as5/10 or greater are used, and a hybridization signal greater thanbackground for one or more of these probes is used to select clones.Adchromosomal plants or parts containing such mini-chromosomes arecontemplated.

The advantages of the present invention include: provision of anautonomous, independent genetic linkage group for accelerating breeding;lack of disruption of host genome; multiple gene “stacking” of largenumbers of genes with a potentially unlimited payload; uniformity ofgenetic composition exogenous DNA sequences in plant cells and plantscontaining autonomous mini-chromosomes; defined genetic context forpredictable gene expression; higher frequency occurrence and recovery ofplant cells and plants containing stably maintained exogenous DNA due toelimination of inefficient integration step; and the ability toeliminate mini-chromosomes in any tissues.

I. Composition of Mini-Chromosomes and Mini-Chromosome Construction

The mini-chromosome vector of the present invention may contain avariety of elements, including (1) sequences that function as plantcentromeres, (2) one or more exogenous nucleic acids, including, forexample, plant-expressed genes, (3) sequences that function as an originof replication, which may be included in the region that functions asplant centromere, (4) optionally, a bacterial plasmid backbone forpropagation of the plasmid in bacteria, (5) optionally, sequences thatfunction as plant telomeres, (6) optionally, additional “stuffer DNA”sequences that serve to separate the various components on themini-chromosome from each other, (7) optionally “buffer” sequences suchas MARs or SARs, (8) optionally marker sequences of any origin,including but not limited to plant and bacterial origin, (9) optionally,sequences that serve as recombination sites, and (10) “chromatinpackaging sequences” such as cohesion and condensing binding sites.

The mini-chromosomes of the present invention may be constructed toinclude various components which are novel, which include, but are notlimited to, the centromere comprising novel repeating centromericsequences, and the promoters, particularly promoters derived fromnon-plant species, as described in further detail below.

The mini-chromosomes of the present invention may be constructed toinclude various components which are novel, which include, but are notlimited to, the centromere comprising novel repeating centromericsequences, and the promoters, particularly promoters derived fromnon-plant species, as described in further detail below.

Novel Centromere Compositions

The centromere in the mini-chromosome of the present invention maycomprise novel repeating centromeric sequences. An example of themini-chromosome in the present invention is the Brassica BB5R4-1mini-chromosome. The sequences set out as SEQ ID NOS:1 to 3 are relevantto the BB5R4-1 mini-chromosomes. The centromere of the BB5R4-1mini-chromosome is 50 kb of Brassica centromere DNA as determined byCHEF gel analysis. To determine the sequence composition of thecentromere, the mini-chromosome was randomly sheared and small fragmentswere cloned for sequencing, from which 11,010 bases of sequence wereobtained from the centromere insert, a 0.17× coverage of the centromere.Of this sequence 9,533 bases were composed of centromere satelliterepeat, the consensus of which is shown in SEQ ID NO:2. The satelliterepeat was found to be 180±2 bp long. The remaining 1,477 bases ofmini-chromosome sequence covered a unique sequence set out as SEQ IDNO:3. This sequence is considered a sampling of the centromere contentof BB5R4-1.

Additional sequence analysis of another sampling of the Brassicacentromere content of BB5R4-1 analyzing 7 contigs (1, 175, 176, 177,180, 184) that contain 118 canrep repeats from BB5R4-1 with repeatlengths of: 113×176 bp, 1×175 bp and 4×174 bp generated the consensussequence set out in SEQ ID NO: 51. A consensus sequence was also builtfrom 135 tandem repeats obtained from another mini-chromosome,BB280R2-3; from the largest contig (33703 kb) spanning a total of 23782bp. The repeat lengths are: 125×176 bp, 4×182 bp, 4×175 bp and 2×177 bpand this sequence is set out as SEQ ID NO: 52. An alignment of SEQ IDNOS: 2, 51 and 52 is set out in FIG. 5.

In another example, individual satellite repeats from soybean BAC cloneSB12R2-3 (SEQ ID NO: 24) showed an average of 91.3% (s.d.=11.3%)identity to each other, with specific regions showing significantlyhigher and lower levels of variability. Comparing the satellite repeatconsensus from SB12R2-3 to that obtained from randomly sampled soybeansatellite sequences ChrGm1 (SEQ ID NO: 25) and ChrGm2 (SEQ ID NO: 26),see U.S. Patent Application 20030124561: Plant centromere compositions)identified several bases that differed significantly (χ² test, P<0.05).The SB12MC satellite repeats showed an average length of 91.07±0.40 bp,similar to the ChrGm2 91-base consensus and differing from the ChrGm192-base consensus. An alignment of the of consensus centromere satelliterepeats is set out in FIG. 6.

Exemplary embodiments of centromere nucleic acid sequences according tothe present invention include fragments or variants of the genomic DNAinserts of the BAC clones [identified as BB5, SB6, TB99, ZB19, or ZB113deposited on Feb. 23, 2005 with the American Type Culture Collection(ATCC), P.O. Box 1549 Manassas, Va. 20108, USA, under Accession Nos.______, ______ and, ______, respectively] that retain the ability tosegregate during mitotic or meiotic division as described herein.Variants of such sequences include artificially produced modificationsas described herein and modifications produced via passaging through oneor more bacterial, plant or other host cells as described herein.

Vectors comprising one, two, three, four, five, six, seven, eight, nine,ten, 15 or 20 or more of the elements contained in any of the exemplaryvectors described in the examples below are also contemplated.

The invention specifically contemplates the alternative use of fragmentsor variants (mutants) of any of the nucleic acids described herein thatretain the desired activity, including nucleic acids that function ascentromeres, nucleic acids that function as promoters or otherregulatory control sequences, or exogenous nucleic acids. Variants mayhave one or more additions, substitutions or deletions of nucleotideswithin the original nucleotide sequence. Variants include nucleic acidsequences that are at least 50%, 55%, 60, 65, 70, 75, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%identical to the original nucleic acid sequence. Variants also includenucleic acid sequences that hybridize under low, medium, high or veryhigh stringency conditions to the original nucleic acid sequence.Similarly, the specification also contemplates the alternative use offragments or variants of any of the polypeptides described herein.

The comparison of sequences and determination of percent identitybetween two nucleotide sequences can be accomplished using amathematical algorithm. In a preferred embodiment, the percent identitybetween two amino acid sequences is determined using the Needleman andWunsch (1970) J. Mol. Biol. 48:444-453 algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix.Preferably parameters are set so as to maximize the percent identity.

As used herein, the term “hybridizes under low stringency, mediumstringency, and high stringency conditions” describes conditions forhybridization and washing. Guidance for performing hybridizationreactions can be found in Current Protocols in Molecular Biology (1989)John Wiley & Sons, N.Y., 6.3.1-6.3.6, which is incorporated byreference. Aqueous and non-aqueous methods are described in thatreference and either can be used. Specific hybridization conditionsreferred to herein are as follows: 1) low stringency hybridizationconditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by two washes in 0.5×SSC, 0.1% SDS, at least at 50C; 2) mediumstringency hybridization conditions in 6×SSC at about 45° C., followedby one or more washes in 0.2×SSC, 0.1% SDS at 55° C.; 3) high stringencyhybridization conditions in 6×SSC at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 65° C.

Mini-Chromosome Sequence Content and Structure

Plant-expressed genes from non-plant sources may be modified toaccommodate plant codon usage, to insert preferred motifs near thetranslation initiation ATG codon, to remove sequences recognized inplants as 5′ or 3′ splice sites, or to better reflect plant GC/ATcontent. Plant genes typically have a GC content of more than 35%, andcoding sequences which are rich in A and T nucleotides can beproblematic. For example, ATTTA motifs may destabilize mRNA; plantpolyadenylation signals such as AATAAA at inappropriate positions withinthe message may cause premature truncation of transcription; andmonocotyledons may recognize AT-rich sequences as splice sites.

Each exogenous nucleic acid or plant-expressed gene may include apromoter, a coding region and a terminator sequence, which may beseparated from each other by restriction endonuclease sites orrecombination sites or both. Genes may also include introns, which maybe present in any number and at any position within the transcribedportion of the gene, including the 5′ untranslated sequence, the codingregion and the 3′ untranslated sequence. Introns may be natural plantintrons derived from any plant, or artificial introns based on thesplice site consensus that has been defined for plant species. Someintron sequences have been shown to enhance expression in plants.Optionally the exogenous nucleic acid may include a planttranscriptional terminator, non-translated leader sequences derived fromviruses that enhance expression, a minimal promoter, or a signalsequence controlling the targeting of gene products to plantcompartments or organelles.

The coding regions of the genes can encode any protein, including butnot limited to visible marker genes (for example, fluorescent proteingenes, other genes conferring a visible phenotype to the plant) or otherscreenable or selectable marker genes (for example, conferringresistance to antibiotics, herbicides or other toxic compounds orencoding a protein that confers a growth advantage to the cellexpressing the protein) or genes which confer some commercial oragronomic value to the adchromosomal plant. Multiple genes can be placedon the same mini-chromosome vector, limited only by the number ofrestriction endonuclease sites or site-specific recombination sitespresent in the vector. The genes may be separated from each other byrestriction endonuclease sites, homing endonuclease sites, recombinationsites or any combinations thereof. Any number of genes can be present.FIGS. 1 and 2 show mini-chromosome vector structures with 2 and 4 genes,respectively.

The mini-chromosome vector may also contain a bacterial plasmid backbonefor propagation of the plasmid in bacteria such as E. coli, A.tumefaciens, or A. rhizogenes. The plasmid backbone may be that of alow-copy vector or in other embodiments it may be desirable to use a midto high level copy backbone. In one embodiment of the invention, thisbackbone contains the replicon of the F′ plasmid of E. coli. However,other plasmid replicons, such as the bacteriophage P1 replicon, or otherlow-copy plasmid systems such as the RK2 replication origin, may also beused. The backbone may include one or several antibiotic-resistancegenes conferring resistance to a specific antibiotic to the bacterialcell in which the plasmid is present. Bacterial antibiotic-resistancegenes include but are not limited to kanamycin-, ampicillin-,chloramphenicol-, streptomycin-, spectinomycin-, tetracycline- andgentamycin-resistance genes.

The mini-chromosome vector may also contain plant telomeres. Anexemplary telomere sequence is TTTAGGG or its complement. Telomeres arespecialized DNA structures at the ends of linear chromosomes thatfunction to stabilize the ends and facilitate the complete replicationof the extreme termini of the DNA molecule (Richards et. al., Cell. Apr.8, 1988;53(1):127-36; Ausubel et al., Current Protocols in MolecularBiology, Wiley & Sons, 1997).

Additionally, the mini-chromosome vector may contain “stuffer DNA”sequences that serve to separate the various components on themini-chromosome (centromere, genes, telomeres) from each other. Thestuffer DNA may be of any origin, prokaryotic or eukaryotic, and fromany genome or species, plant, animal, microbe or organelle, or may be ofsynthetic origin. The stuffer DNA can range from 100 bp to 10 Mb inlength and can be repetitive in sequence, with unit repeats from 10 to1,000,000 bp. Examples of repetitive sequences that can be used asstuffer DNAs include but are not limited to: rDNA, satellite repeats,retroelements, transposons, pseudogenes, transcribed genes,microsatellites, tDNA genes, short sequence repeats and combinationsthereof. Alternatively, the stuffer DNA can consist of unique,non-repetitive DNA of any origin or sequence. The stuffer sequences mayalso include DNA with the ability to form boundary domains, such as butnot limited to scaffold attachment regions (SARs) or matrix attachmentregions (MARs). The stuffer DNA may be entirely synthetic, composed ofrandom sequence. In this case, the stuffer DNA may have any basecomposition, or any A/T or G/C content. For example, the G/C content ofthe stuffer DNA could resemble that of the plant (˜30-40%), or could bemuch lower (0-30%) or much higher (40-100%). Alternatively, the stuffersequences could be synthesized to contain an excess of any givennucleotide such as A, C, G or T. Different synthetic stuffers ofdifferent compositions may also be combined with each other. For examplea fragment with low G/C content may be flanked or abutted by a fragmentof medium or high G/C content, or vice versa.

In one embodiment of the invention, the mini-chromosome has a circularstructure without telomeres, as shown in FIGS. 1 and 2 “circular”. Inanother embodiment, the mini-chromosome has a circular structure withtelomeres, as shown in FIGS. 1 and 2 “linear”. In a third embodiment,the mini-chromosome has a linear structure with telomeres, as wouldresult if the “linear” structure shown in FIGS. 1 and 2 were to be cutwith a unique endonuclease, exposing the telomeres at the ends of a DNAmolecule that contains all of the sequence contained in the original,closed construct with the exception of the antibiotic-resistance gene#1. In a fourth embodiment of the invention, the telomeres could beplaced in such a manner that the bacterial replicon, backbone sequences,antibiotic-resistance genes and any other sequences of bacterial originand present for the purposes of propagation of the mini-chromosome inbacteria, can be removed from the plant-expressed genes, the centromere,telomeres, and other sequences by cutting the structure with uniqueendonuclease #2 (FIG. 3). This results in a mini-chromosome from whichmuch of, or preferably all, bacterial sequences have been removed. Inthis embodiment, bacterial sequence present between or among theplant-expressed genes or other mini-chromosome sequences would beexcised prior to removal of the remaining bacterial sequences by cuttingthe mini-chromosome with homing endonuclease #1, and re-ligating thestructure such that the antibiotic-resistance gene #1 has been lost(FIG. 3). In all of the structures shown in FIGS. 1, 2 and 3, the uniqueendonuclease site may be the recognition sequence of a homingendonuclease. Alternatively, the endonucleases and their sites can bereplaced with any specific DNA cutting mechanism and its specificrecognition site such as rare-cutting endonuclease or recombinase andits specific recognition site, as long as that site is present in themini-chromosomes only at the indicated positions.

Various structural configurations are possible by which mini-chromosomeelements can be oriented with respect to each other. A centromere can beplaced on a mini-chromosome either between genes or outside a cluster ofgenes next to one telomere or next to the other telomere. Stuffer DNAscan be combined with these configurations to place the stuffer sequencesinside the telomeres, around the centromere between genes or anycombination thereof. Thus, a large number of alternative mini-chromosomestructures are possible, depending on the relative placement ofcentromere DNA, genes, stuffer DNAs, bacterial sequences, telomeres, andother sequences. The sequence content of each of these variants is thesame, but their structure may be different depending on how thesequences are placed. These variations in architecture are possible bothfor linear and for circular mini-chromosomes.

Exemplary Centromere Components

Centromere components may be isolated or derived from native plantgenome, for example, modified through recombinant techniques or throughthe cell-based techniques described below. Alternatively, whollyartificial centromere components may be constructed using as a generalguide the sequence of native centromeres. Combinations of centromerecomponents derived from natural sources and/or combinations of naturallyderived and artificial components are also contemplated As noted above,centromere sequence from one taxonomic plant species has been shown tobe functional in another taxonomic plant species, genus and family.

In one embodiment, the centromere contains n copies of a repeatednucleotide sequence obtained by the methods disclosed herein, wherein nis at least 2. In another embodiment, the centromere contains n copiesof interdigitated repeats. An interdigitated repeat is a DNA sequencethat consists of two distinct repetitive elements that combine to createan unique permutation. Potentially any number of repeat copies capableof physically being placed on the recombinant construct could beincluded on the construct, including about 5, 10, 15, 20, 30, 50, 75,100, 150, 200, 300, 400, 500, 750, 1,000, 1,500, 2,000, 3,000, 5,000,7,500, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000,90,000 and about 100,000, including all ranges in-between such copynumbers. Moreover, the copies, while largely identical, can vary fromeach other. Such repeat variation is commonly observed in naturallyoccurring centromeres. The length of the repeat may vary, but willpreferably range from about 20 bp to about 360 bp, from about 20 bp toabout 250 bp, from about 50 bp to about 225 bp, from about 75 bp toabout 210 bp, such as a 92 bp repeat and a 97 bp repeat, from about 100bp to about 205 bp, from about 125 bp to about 200 bp, from about 150 bpto about 195 bp, from about 160 bp to about 190 and from about 170 bp toabout 185 bp including about 180 bp.

Modification of Centromeres Isolated from Native Plant Genome

Modification and changes may be made in the centromeric DNA segments ofthe current invention and still obtain a functional molecule withdesirable characteristics. The following is a discussion based uponchanging the nucleic acids of a centromere to create an equivalent, oreven an improved, second generation molecule.

In particular embodiments of the invention, mutated centromericsequences are contemplated to be useful for increasing the utility ofthe centromere. It is specifically contemplated that the function of thecentromeres of the current invention may be based in part of in wholeupon the secondary structure of the DNA sequences of the centromere,modification of the DNA with methyl groups or other adducts, and/or theproteins which interact with the centromere. By changing the DNAsequence of the centromere, one may alter the affinity of one or morecentromere-associated protein(s) for the centromere and/or the secondarystructure or modification of the centromeric sequences, thereby changingthe activity of the centromere. Alternatively, changes may be made inthe centromeres of the invention which do not affect the activity of thecentromere. Changes in the centromeric sequences which reduce the sizeof the DNA segment needed to confer centromere activity are contemplatedto be particularly useful in the current invention, as would changeswhich increased the fidelity with which the centromere was transmittedduring mitosis and meiosis.

Modification of Centromeres by Passage through Bacteria, Plant or OtherHosts or Processes

In the methods of the present invention, the resulting mini-chromosomeDNA sequence may also be a derivative of the parental clone orcentromere clone having substitutions, deletions, insertions,duplications and/or rearrangements of one or more nucleotides in thenucleic acid sequence. Such nucleotide mutations may occur individuallyor consecutively in stretches of 1, 2, 3, 4, 5, 10, 20, 40, 80, 100,200, 400, 800, 1000, 2000, 4000, 8000, 10000, 50000, 100000, and about200000, including all ranges in-between.

Variations of mini-chromosomes may arise through passage ofmini-chromosomes through various hosts including virus, bacteria, yeast,plant or other prokaryotic or eukaryotic organism and may occur throughpassage of multiple hosts or individual host. Variations may also occurby replicating the mini-chromosome in vitro.

Derivatives may be identified through sequence analysis, or variationsin mini-chromosome molecular weight through electrophoresis such as, butnot limited to, CHEF gel analysis, column or gradient separation, or anyother methods used in the field to determine and/or analyze DNAmolecular weight or sequence content. Alternately, derivatives may beidentified by the altered activity of a derivative in conferringcentromere function to a mini-chromosome.

Exemplary Exogenous Nucleic Acids Including Plant-Expressed Genes

Of particular interest in the present invention are exogenous nucleicacids which when introduced into plants alter the phenotype of theplant, a plant organ, plant tissue, or portion of the plant. Exemplaryexogenous nucleic acids encode polypeptides involved in one or moreimportant biological properties in plants. Other exemplary exogenousnucleic acids alter expression of exogenous or endogenous genes, eitherincreasing or decreasing expression, optionally in response to aspecific signal or stimulus.

As used herein, the term “trait” can refer either to the alteredphenotype of interest or the nucleic acid which causes the alteredphenotype of interest.

One of the major purposes of transformation of crop plants is to addsome commercially desirable, agronomically important traits to theplant. Such traits include, but are not limited to, herbicide resistanceor tolerance; insect (pest) resistance or tolerance; disease resistanceor tolerance (viral, bacterial, fungal, nematode or other pathogens);stress tolerance and/or resistance, as exemplified by resistance ortolerance to drought, heat, chilling, freezing, excessive moisture, saltstress, mechanical stress, extreme acidity, alkalinity, toxins, UVlight, ionizing radiation or oxidative stress; increased yields, whetherin quantity or quality; enhanced or altered nutrient acquisition andenhanced or altered metabolic efficiency; enhanced or alterednutritional content and makeup of plant tissues used for food, feed,fiber or processing; physical appearance; male sterility; drydown;standability; prolificacy; starch quantity and quality; oil quantity andquality; protein quality and quantity; amino acid composition; modifiedchemical production; altered pharmaceutical or nutraceutical properties;altered bioremediation properties; increased biomass; altered growthrate; altered fitness; altered biodegradability; altered CO₂ fixation;presence of bioindicator activity; altered digestibility by humans oranimals; altered allergenicity; altered mating characteristics; alteredpollen dispersal; improved environmental impact; altered nitrogenfixation capability; the production of a pharmaceutically activeprotein; the production of a small molecule with medicinal properties;the production of a chemical including those with industrial utility;the production of nutraceuticals, food additives, carbohydrates, RNAs,lipids, fuels, dyes, pigments, vitamins, scents, flavors, vaccines,antibodies, hormones, and the like; and alterations in plantarchitecture or development, including changes in developmental timing,photosynthesis, signal transduction, cell growth, reproduction, ordifferentiation. Additionally one could create a library of an entiregenome from any organism or organelle including mammals, plants,microbes, fungi, or bacteria, represented on mini-chromosomes.

In one embodiment, the modified plant may exhibit increased or decreasedexpression or accumulation of a product of the plant, which may be anatural product of the plant or a new or altered product of the plant.Exemplary products include an enzyme, an RNA molecule, a nutritionalprotein, a structural protein, an amino acid, a lipid, a fatty acid, apolysaccharide, a sugar, an alcohol, an alkaloid, a carotenoid, apropanoid, a phenylpropanoid, or terpenoid, a steroid, a flavonoid, aphenolic compound, an anthocyanin, a pigment, a vitamin or a planthormone. In another embodiment, the modified plant has enhanced ordiminished requirement for light, water, nitrogen, or trace elements. Inanother embodiment the modified plant has an enhance ability to captureor fix nitrogen from its environment. In yet another embodiment, themodified plant is enriched for an essential amino acid as a proportionof a protein fraction of the plant. The protein fraction may be, forexample, total seed protein, soluble protein, insoluble protein,water-extractable protein, and lipid-associated protein. Themodification may include overexpression, underexpression, antisensemodulation, sense suppression, inducible expression, induciblerepression, or inducible modulation of a gene.

A brief summary of exemplary improved properties and polypeptides ofinterest for either increased or decreased expression is provided below.

(i) Herbicide Resistance

A herbicide resistance (or tolerance) trait is a characteristic of amodified plant that is resistant to dosages of an herbicide that istypically lethal to a non-modified plant. Exemplary herbicides for whichresistance is useful in a plant include glyphosate herbicides,phosphinothricin herbicides, oxynil herbicides, imidazolinoneherbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylureaherbicides, bialaphos herbicides, sulfonamide herbicides and glufosinateherbicides. Other herbicides would be useful as would combinations ofherbicide genes on the same mini-chromosome.

The genes encoding phosphinothricin acetyltransferase (bar), glyphosatetolerant EPSP synthase genes, glyphosate acetyltransferase, theglyphosate degradative enzyme gene gox encoding glyphosateoxidoreductase, deh (encoding a dehalogenase enzyme that inactivatesdalapon), herbicide resistant (e.g., sulfonylurea and imidazolinone)acetolactate synthase, and bxn genes (encoding a nitrilase enzyme thatdegrades bromoxynil) are good examples of herbicide resistant genes foruse in transformation. The bar gene codes for an enzyme,phosphinothricin acetyltransferase (PAT), which inactivates theherbicide phosphinothricin and prevents this compound from inhibitingglutamine synthetase enzymes. The enzyme 5 enolpyruvylshikimate 3phosphate synthase (EPSP Synthase), is normally inhibited by theherbicide N (phosphonomethylglycine (glyphosate). However, genes areknown that encode glyphosate resistant EPSP synthase enzymes. Thesegenes are particularly contemplated for use in plant transformation. Thedeh gene encodes the enzyme dalapon dehalogenase and confers resistanceto the herbicide dalapon. The bxn gene codes for a specific nitrilaseenzyme that converts bromoxynil to a non herbicidal degradation product.The glyphosate acetyl transferase gene inactivates the herbicideglyphosate and prevents this compound from inhibiting EPSP synthase.

Polypeptides that may produce plants having tolerance to plantherbicides include polypeptides involved in the shikimate pathway, whichare of interest for providing glyphosate tolerant plants. Suchpolypeptides include polypeptides involved in biosynthesis ofchorismate, phenylalanine, tyrosine and tryptophan.

(ii) Insect Resistance

Potential insect resistance (or tolerance) genes that can be introducedinclude Bacillus thuringensis crystal toxin genes or Bt genes (Watrud etal., In: Engineered Organisms and the Environment, 1985). Bt genes mayprovide resistance to lepidopteran or coleopteran pests such as EuropeanCorn Borer (ECB). Preferred Bt toxin genes for use in such embodimentsinclude the CryIA(b) and CryIA(c) genes. Endotoxin genes from otherspecies of B. thuringensis which affect insect growth or developmentalso may be employed in this regard.

It is contemplated that preferred Bt genes for use in themini-chromosomes disclosed herein will be those in which the codingsequence has been modified to effect increased expression in plants, andfor example, in monocot plants. Means for preparing synthetic genes arewell known in the art and are disclosed in, for example, U.S. Pat. No.5,500,365 and U.S. Pat. No. 5,689,052, each of the disclosures of whichare specifically incorporated herein by reference in their entirety.Examples of such modified Bt toxin genes include a synthetic Bt CryIA(b)gene (Perlak et al., Proc. Natl. Acad. Sci. USA, 88:3324-3328, 1991),and the synthetic CryIA(c) gene termed 1800b (PCT Application WO95/06128). Some examples of other Bt toxin genes known to those of skillin the art are given in Table 1 below. TABLE 1 Bacillus thuringiensisEndotoxin Genes^(a) New Nomenclature Old Nomenclature GenBank AccessionCry1Aa CryIA(a) M11250 Cry1Ab CryIA(b) M13898 Cry1Ac CryIA(c) M11068Cry1Ad CryIA(d) M73250 Cry1Ae CryIA(e) M65252 Cry1Ba CryIB X06711 Cry1BbET5 L32020 Cry1Bc PEG5 Z46442 Cry1Bd CryE1 U70726 Cry1Ca CryIC X07518Cry1Cb CryIC(b) M97880 Cry1Da CryID X54160 Cry1Db PrtB Z22511 Cry1EaCryIE X53985 Cry1Eb CryIE(b) M73253 Cry1Fa CryIF M63897 Cry1Fb PrtDZ22512 Cry1Ga PrtA Z22510 Cry1Gb CryH2 U70725 Cry1Ha PrtC Z22513 Cry1HbU35780 Cry1Ia CryV X62821 Cry1Ib CryV U07642 Cry1Ja ET4 L32019 Cry1JbET1 U31527 Cry1K U28801 Cry2Aa CryIIA M31738 Cry2Ab CryIIB M23724 Cry2AcCryIIC X57252 Cry3A CryIIIA M22472 Cry3Ba CryIIIB X17123 Cry3Bb CryIIIB2M89794 Cry3C CryIIID X59797 Cry4A CryIVA Y00423 Cry4B CryIVB X07423Cry5Aa CryVA(a) L07025 Cry5Ab CryVA(b) L07026 Cry6A CryVIA L07022 Cry6BCryVIB L07024 Cry7Aa CryIIIC M64478 Cry7Ab CryIIICb U04367 Cry8A CryIIIEU04364 Cry8B CryIIIG U04365 Cry8C CryIIIF U04366 Cry9A CryIG X58120Cry9B CryIX X75019 Cry9C CryIH Z37527 Cry10A CryIVC M12662 Cry11A CryIVDM31737 Cry11B Jeg80 X86902 Cry12A CryVB L07027 Cry13A CryVC L07023Cry14A CryVD U13955 Cry15A 34 kDa M76442 Cry16A cbm71 X94146 Cry17Acbm71 X99478 Cry18A CryBP1 X99049 Cry19A Jeg65 Y08920 Cyt1Aa CytA X03182Cyt1Ab CytM X98793 Cyt2A CytB Z14147 Cyt2B CytB U52043^(a)Adapted from:http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html

Protease inhibitors also may provide insect resistance (Johnson et al.,Proc Natl Acad Sci USA. 1989 December; 86(24): 9871-9875.), and willthus have utility in plant transformation. The use of a proteaseinhibitor II gene, pinII, from tomato or potato is envisioned to beparticularly useful. Even more advantageous is the use of a pinII genein combination with a Bt toxin gene, the combined effect of which hasbeen discovered to produce synergistic insecticidal activity. Othergenes which encode inhibitors of the insect's digestive system, or thosethat encode enzymes or co factors that facilitate the production ofinhibitors, also may be useful. This group may be exemplified byoryzacystatin and amylase inhibitors such as those from wheat andbarley.

Amylase inhibitors are found in various plant species and are used toward off insect predation via inhibition of the digestive amylases ofattacking insects. Several amylase inhibitor genes have been isolatedfrom plants and some have been introduced as exogenous nucleic acids,conferring an insect resistant phenotype that is potentially useful(“Plants, Genes, and Crop Biotechnology” by Maarten J. Chrispeels andDavid E. Sadava (2003) Jones and Bartlett Press).

Genes encoding lectins may confer additional or alternative insecticideproperties. Lectins are multivalent carbohydrate binding proteins whichhave the ability to agglutinate red blood cells from a range of species.Lectins have been identified recently as insecticidal agents withactivity against weevils, ECB and rootworm (Murdock et al.,Phytochemistry, 29:85-89, 1990, Czapla & Lang, J. Econ. Entomol.,83:2480-2485, 1990). Lectin genes contemplated to be useful include, forexample, barley and wheat germ agglutinin (WGA) and rice lectins(Gatehouse et al., J. Sci. Food. Agric., 35.373-380, 1984), with WGAbeing preferred.

Genes controlling the production of large or small polypeptides activeagainst insects when introduced into the insect pests, such as, e.g.,lytic peptides, peptide hormones and toxins and venoms, form anotheraspect of the invention. For example, it is contemplated that theexpression of juvenile hormone esterase, directed towards specificinsect pests, also may result in insecticidal activity, or perhaps causecessation of metamorphosis (Hammock et al., Nature, 344:458-461, 1990).

Genes which encode enzymes that affect the integrity of the insectcuticle form yet another aspect of the invention. Such genes includethose encoding, e.g., chitinase, proteases, lipases and also genes forthe production of nikkomycin, a compound that inhibits chitin synthesis,the introduction of any of which is contemplated to produce insectresistant plants. Genes that code for activities that affect insectmolting, such as those affecting the production of ecdysteroid UDPglucosyl transferase, also fall within the scope of the useful exogenousnucleic acids of the present invention.

Genes that code for enzymes that facilitate the production of compoundsthat reduce the nutritional quality of the host plant to insect pestsalso are encompassed by the present invention. It may be possible, forinstance, to confer insecticidal activity on a plant by altering itssterol composition. Sterols are obtained by insects from their diet andare used for hormone synthesis and membrane stability. Thereforealterations in plant sterol composition by expression of novel genes,e.g., those that directly promote the production of undesirable sterolsor those that convert desirable sterols into undesirable forms couldhave a negative effect on insect growth and/or development and henceendow the plant with insecticidal activity. Lipoxygenases are naturallyoccurring plant enzymes that have been shown to exhibit anti nutritionaleffects on insects and to reduce the nutritional quality of their diet.Therefore, further embodiments of the invention concern modified plantswith enhanced lipoxygenase activity which may be resistant to insectfeeding.

Tripsacum dactyloides is a species of grass that is resistant to certaininsects, including corn root worm. It is anticipated that genes encodingproteins that are toxic to insects or are involved in the biosynthesisof compounds toxic to insects will be isolated from Tripsacum and thatthese novel genes will be useful in conferring resistance to insects. Itis known that the basis of insect resistance in Tripsacum is genetic,because said resistance has been transferred to Zea mays via sexualcrosses (Branson and Guss, Proceedings North Central BranchEntomological Society of America, 27:91-95, 1972). It is furtheranticipated that other cereal, monocot or dicot plant species may havegenes encoding proteins that are toxic to insects which would be usefulfor producing insect resistant plants.

Further genes encoding proteins characterized as having potentialinsecticidal activity also may be used as exogenous nucleic acids inaccordance herewith. Such genes include, for example, the cowpea trypsininhibitor (CpTI; Hilder et al., Nature, 330:160-163, 1987) which may beused as a rootworm deterrent; genes encoding avermectin (Avermectin andAbamectin., Campbell, W. C., Ed., 1989; Ikeda et al., J. Bacteriol.,169:5615-5621, 1987) which may prove particularly useful as a cornrootworm deterrent; ribosome inactivating protein genes; and even genesthat regulate plant structures. Modified plants including anti insectantibody genes and genes that code for enzymes that can convert a nontoxic insecticide (pro insecticide) applied to the outside of the plantinto an insecticide inside the plant also are contemplated.

Polypeptides that may improve plant tolerance to effects of plant pestsor pathogens include proteases, polypeptides involved in anthocyaninbiosynthesis, polypeptides involved in cell wall metabolism, includingcellulases, glucosidases, pectin methylesterase, pectinase,polygalacturonase, chitinase, chitosanase, and cellulose synthase, andpolypeptides involved in biosynthesis of terpenoids or indole forproduction of bioactive metabolites to provide defense againstherbivorous insects. It is also anticipated that combinations ofdifferent insect resistance genes on the same mini-chromosome will beparticularly useful.

Vegetative Insecticidal Proteins (VIP) are a relatively new class ofproteins originally found to be produced in the vegetative growth phaseof the bacterium, Bacillus cereus, but do have a spectrum of insectlethality similar to the insecticidal genes found in strains of Bacillusthuringensis. Both the vip1a and vip3A genes have been isolated and havedemonstrated insect toxicity. It is anticipated that such genes may beused in modified plants to confer insect resistance (“Plants, Genes, andCrop Biotechnology” by Maarten J. Chrispeels and David E. Sadava (2003)Jones and Bartlett Press).

(iii) Environment or Stress Resistance

Improvement of a plant's ability to tolerate various environmentalstresses such as, but not limited to, drought, excess moisture,chilling, freezing, high temperature, salt, and oxidative stress, alsocan be effected through expression of novel genes. It is proposed thatbenefits may be realized in terms of increased resistance to freezingtemperatures through the introduction of an “antifreeze” protein such asthat of the Winter Flounder (Cutler et al., J. Plant Physiol.,135:351-354, 1989) or synthetic gene derivatives thereof. Improvedchilling tolerance also may be conferred through increased expression ofglycerol 3 phosphate acetyltransferase in chloroplasts (Wolter et al.,The EMBO J., 4685-4692, 1992). Resistance to oxidative stress (oftenexacerbated by conditions such as chilling temperatures in combinationwith high light intensities) can be conferred by expression ofsuperoxide dismutase (Gupta et al., 1993), and may be improved byglutathione reductase (Bowler et al., Ann Rev. Plant Physiol.,43:83-116, 1992). Such strategies may allow for tolerance to freezing innewly emerged fields as well as extending later maturity higher yieldingvarieties to earlier relative maturity zones.

It is contemplated that the expression of novel genes that favorablyaffect plant water content, total water potential, osmotic potential, orturgor will enhance the ability of the plant to tolerate drought. Asused herein, the terms “drought resistance” and “drought tolerance” areused to refer to a plant's increased resistance or tolerance to stressinduced by a reduction in water availability, as compared to normalcircumstances, and the ability of the plant to function and survive inlower water environments. In this aspect of the invention it isproposed, for example, that the expression of genes encoding for thebiosynthesis of osmotically active solutes, such as polyol compounds,may impart protection against drought. Within this class are genesencoding for mannitol L phosphate dehydrogenase (Lee and Saier, 1982)and trehalose 6 phosphate synthase (Kaasen et al., J. Bacteriology,174:889-898, 1992). Through the subsequent action of native phosphatasesin the cell or by the introduction and coexpression of a specificphosphatase, these introduced genes will result in the accumulation ofeither mannitol or trehalose, respectively, both of which have been welldocumented as protective compounds able to mitigate the effects ofstress. Mannitol accumulation in transgenic tobacco has been verifiedand preliminary results indicate that plants expressing high levels ofthis metabolite are able to tolerate an applied osmotic stress(Tarczynski et al., Science, 259:508-510, 1993, Tarczynski et al Proc.Natl. Acad. Sci. USA, 89:1-5, 1993).

Similarly, the efficacy of other metabolites in protecting either enzymefunction (e.g., alanopine or propionic acid) or membrane integrity(e.g., alanopine) has been documented (Loomis et al., J. Expt. Zoology,252:9-15, 1989), and therefore expression of genes encoding for thebiosynthesis of these compounds might confer drought resistance in amanner similar to or complimentary to mannitol. Other examples ofnaturally occurring metabolites that are osmotically active and/orprovide some direct protective effect during drought and/or desiccationinclude fructose, erythritol (Coxson et al., Biotropica, 24:121-133,1992), sorbitol, dulcitol (Karsten et al., Botanica Marina, 35:11-19,1992), glucosyiglycerol (Reed et al., J. Gen. Microbiology, 130:1-4,1984; Erdmann et al., J. Gen. Microbiology, 138:363-368, 1992), sucrose,stachyose (Koster and Leopold, Plant Physiol., 88:829-832, 1988;Blackman et al., Plant Physiol., 100:225-230, 1992), raffinose (BernalLugo and Leopold, Plant Physiol., 98:1207-1210, 1992), proline (Rensburget al., J. Plant Physiol., 141:188-194, 1993), glycine betaine, ononitoland pinitol (Vernon and Bohnert, The EMBO J., 11:2077-2085, 1992).Continued canopy growth and increased reproductive fitness during timesof stress will be augmented by introduction and expression of genes suchas those controlling the osmotically active compounds discussed aboveand other such compounds. Currently preferred genes which promote thesynthesis of an osmotically active polyol compound are genes whichencode the enzymes mannitol 1 phosphate dehydrogenase, trehalose 6phosphate synthase and myoinositol 0 methyltransferase.

It is contemplated that the expression of specific proteins also mayincrease drought tolerance. Three classes of Late Embryogenic Proteinshave been assigned based on structural similarities (see Dure et al.,Plant Molecular Biology, 12:475-486, 1989). All three classes of LEAshave been demonstrated in maturing (e.g. desiccating) seeds. Withinthese 3 types of LEA proteins, the Type II (dehydrin type) havegenerally been implicated in drought and/or desiccation tolerance invegetative plant parts (e.g. Mundy and Chua, The EMBO J., 7:2279-2286,1988; Piatkowski et al., Plant Physiol., 94:1682-1688, 1990; YamaguchiShinozaki et al., Plant Cell Physiol., 33:217-224, 1992). Expression ofa Type III LEA (HVA 1) in tobacco was found to influence plant height,maturity and drought tolerance (Fitzpatrick, Gen. Engineering News,22:7, 1993). In rice, expression of the HVA 1 gene influenced toleranceto water deficit and salinity (Xu et al., Plant Physiol., 110:249-257,1996). Expression of structural genes from any of the three LEA groupsmay therefore confer drought tolerance. Other types of proteins inducedduring water stress include thiol proteases, aldolases or transmembranetransporters (Guerrero et al., Plant Molecular Biology, 15:11-26, 1990),which may confer various protective and/or repair type functions duringdrought stress. It also is contemplated that genes that effect lipidbiosynthesis and hence membrane composition might also be useful inconferring drought resistance on the plant.

Many of these genes for improving drought resistance have complementarymodes of action. Thus, it is envisaged that combinations of these genesmight have additive and/or synergistic effects in improving droughtresistance in plants. Many of these genes also improve freezingtolerance (or resistance); the physical stresses incurred duringfreezing and drought are similar in nature and may be mitigated insimilar fashion. Benefit may be conferred via constitutive expression ofthese genes, but the preferred means of expressing these novel genes maybe through the use of a turgor induced promoter (such as the promotersfor the turgor induced genes described in Guerrero et al., PlantMolecular Biology, 15:11-26, 1990 and Shagan et al., Plant Physiol.,101:1397-1398, 1993 which are incorporated herein by reference). Spatialand temporal expression patterns of these genes may enable plants tobetter withstand stress.

It is proposed that expression of genes that are involved with specificmorphological traits that allow for increased water extractions fromdrying soil would be of benefit. For example, introduction andexpression of genes that alter root characteristics may enhance wateruptake. It also is contemplated that expression of genes that enhancereproductive fitness during times of stress would be of significantvalue. For example, expression of genes that improve the synchrony ofpollen shed and receptiveness of the female flower parts, e.g., silks,would be of benefit. In addition it is proposed that expression of genesthat minimize kernel abortion during times of stress would increase theamount of grain to be harvested and hence be of value.

Given the overall role of water in determining yield, it is contemplatedthat enabling plants to utilize water more efficiently, through theintroduction and expression of novel genes, will improve overallperformance even when soil water availability is not limiting. Byintroducing genes that improve the ability of plants to maximize waterusage across a full range of stresses relating to water availability,yield stability or consistency of yield performance may be realized.

Polypeptides that may improve stress tolerance under a variety of stressconditions include polypeptides involved in gene regulation, such asserine/threonine-protein kinases, MAP kinases, MAP kinase kinases, andMAP kinase kinase kinases; polypeptides that act as receptors for signaltransduction and regulation, such as receptor protein kinases;intracellular signaling proteins, such as protein phosphatases, GTPbinding proteins, and phospholipid signaling proteins; polypeptidesinvolved in arginine biosynthesis; polypeptides involved in ATPmetabolism, including for example ATPase, adenylate transporters, andpolypeptides involved in ATP synthesis and transport; polypeptidesinvolved in glycine betaine, jasmonic acid, flavonoid or steroidbiosynthesis; and hemoglobin. Enhanced or reduced activity of suchpolypeptides in modified plants will provide changes in the ability of aplant to respond to a variety of environmental stresses, such aschemical stress, drought stress and pest stress.

Other polypeptides that may improve plant tolerance to cold or freezingtemperatures include polypeptides involved in biosynthesis of trehaloseor raffinose, polypeptides encoded by cold induced genes, fatty acyldesaturases and other polypeptides involved in glycerolipid or membranelipid biosynthesis, which find use in modification of membrane fattyacid composition, alternative oxidase, calcium-dependent proteinkinases, LEA proteins or uncoupling protein.

Other polypeptides that may improve plant tolerance to heat includepolypeptides involved in biosynthesis of trehalose, polypeptidesinvolved in glycerolipid biosynthesis or membrane lipid metabolism (foraltering membrane fatty acid composition), heat shock proteins ormitochondrial NDK.

Other polypeptides that may improve tolerance to extreme osmoticconditions include polypeptides involved in proline biosynthesis.

Other polypeptides that may improve plant tolerance to droughtconditions include aquaporins, polypeptides involved in biosynthesis oftrehalose or wax, LEA proteins or invertase.

(iv) Disease Resistance

It is proposed that increased resistance (or tolerance) to diseases maybe realized through introduction of genes into plants, for example, intomonocotyledonous plants such as maize. It is possible to produceresistance to diseases caused by viruses, viroids, bacteria, fungi andnematodes. It also is contemplated that control of mycotoxin producingorganisms may be realized through expression of introduced genes.Resistance can be effected through suppression of endogenous factorsthat encourage disease-causing interactions, expression of exogenousfactors that are toxic to or otherwise provide protection frompathogens, or expression of factors that enhance the plant's own defenseresponses.

Resistance to viruses may be produced through expression of novel genes.For example, it has been demonstrated that expression of a viral coatprotein in a modified plant can impart resistance to infection of theplant by that virus and perhaps other closely related viruses (Cuozzo etal., Bio/Technology, 6:549-553, 1988, Hemenway et al., The EMBO J.,7:1273-1280, 1988, Abel et al., Science, 232:738-743, 1986). It iscontemplated that expression of antisense genes targeted at essentialviral functions may also impart resistance to viruses. For example, anantisense gene targeted at the gene responsible for replication of viralnucleic acid may inhibit replication and lead to resistance to thevirus. It is believed that interference with other viral functionsthrough the use of antisense genes also may increase resistance toviruses. Further, it is proposed that it may be possible to achieveresistance to viruses through other approaches, including, but notlimited to the use of satellite viruses.

It is proposed that increased resistance to diseases caused by bacteriaand fungi may be realized through introduction of novel genes. It iscontemplated that genes encoding so called “peptide antibiotics,”pathogenesis related (PR) proteins, toxin resistance, or proteinsaffecting host pathogen interactions such as morphologicalcharacteristics will be useful. Peptide antibiotics are polypeptidesequences which are inhibitory to growth of bacteria and othermicroorganisms. For example, the classes of peptides referred to ascecropins and magainins inhibit growth of many species of bacteria andfungi. It is proposed that expression of PR proteins in plants, forexample, monocots such as maize, may be useful in conferring resistanceto bacterial disease. These genes are induced following pathogen attackon a host plant and have been divided into at least five classes ofproteins (Bol, Linthorst, and Cornelissen, 1990). Included amongst thePR proteins are beta 1, 3 glucanases, chitinases, and osmotin and otherproteins that are believed to function in plant resistance to diseaseorganisms. Other genes have been identified that have antifungalproperties, e.g., UDA (stinging nettle lectin), or hevein (Broakaert etal., 1989; Barkai Golan et al., 1978). It is known that certain plantdiseases are caused by the production of phytotoxins. It is proposedthat resistance to these diseases would be achieved through expressionof a novel gene that encodes an enzyme capable of degrading or otherwiseinactivating the phytotoxin. It also is contemplated that expression ofnovel genes that alter the interactions between the host plant andpathogen may be useful in reducing the ability of the disease organismto invade the tissues of the host plant, e.g., an increase in thewaxiness of the leaf cuticle or other morphological characteristics.

Polypeptides useful for imparting improved disease responses to plantsinclude polypeptides encoded by cercosporin induced genes, antifungalproteins and proteins encoded by R-genes or SAR genes.

Agronomically important diseases caused by fungal phytopathogensinclude: glume or leaf blotch, late blight, stalk/head rot, rice blast,leaf blight and spot, corn smut, wilt, sheath blight, stem canker, rootrot, blackleg or kernel rot.

Exemplary plant viruses include tobacco or cucumber mosaic virus,ringspot virus, necrosis virus, maize dwarf mosaic virus, etc. Specificfungal, bacterial and viral pathogens of major crops include, but arenot limited to:

RICE: rice brown spot fungus (Cochliobolus miyabeanus), rice blastfungus—Magnaporthe grisea (Pyricularia grisea), Magnaporthe salvinii(Sclerotium oryzae), Xanthomomas oryzae pv. oryzae, Xanthomomas oryzaepv. oryzicola, Rhizoctonia spp. (including but not limited toRhizoctonia solani, Rhizoctonia oryzae and Rhizoctonia oryzae-sativae),Pseudomonas spp. (including but not limited to Pseudomonas plantarii,Pseudomonas avenae, Pseudomonas glumae, Pseudomonas fuscovaginae,Pseudomonas alboprecipitans, Pseudomonas syringae pv. panici,Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. oryzae andPseudomonas syringae pv. aptata), Erwinia spp. (including but notlimited to Erwinia herbicola, Erwinia amylovaora, Erwinia chrysanthemiand Erwinia carotovora), Achyla spp. (including but not limited toAchyla conspicua and Achyia klebsiana), Pythium spp. (including but notlimited to Pythium dissotocum, Pythium irregulare, Pythium arrhenomanes,Pythium myriotylum, Pythium catenulatum, Pythium graminicola and Pythiumspinosum), Saprolegnia spp., Dictyuchus spp., Pythiogeton spp.,Phytophthora spp., Alternaria padwickii, Cochliobolus miyabeanus,Curvularia spp. (including but not limited to Curvularia lunata,Curvularia affinis, Curvularia clavata, Curvularia eragrostidis,Curvularia fallax, Curvularia geniculata, Curvularia inaequalis,Curvularia intermedia, Curvularia oryzae, Curvularia oryzae-sativae,Curvularia pallescens, Curvularia senegalensis, Curvularia tuberculata,Curvularia uncinata and Curvularia verruculosa), Sarocladium oryzae,Gerlachia oryzae, Fusarium spp. (including but not limited Fusariumgraminearum, Fusarium nivale and to different pathovars of Fusariummonolifonne, including pvs. fujikuroi and zeae), Sclerotium rolfsii,Phoma exigua, Mucor fragilis, Trichoderma viride, Rhizopus spp.,Cercospora oryzae, Entyloma oryzae, Dreschlera gigantean, Scierophthoramacrospora, Mycovellosiella oryzae, Phomopsis oryzae-sativae, Pucciniagraminis, Uromyces coronatus, Cylindrocladium scoparium, Sarocladiumoryzae, Gaeumannomyces graminis pv. graminis, Myrothecium verrucaria,Pyrenochaeta oryzae, Ustilaginoidea virens, Neovossia spp. (includingbut not limited to Neovossia horrida), Tilletia spp., Balansiaoryzae-sativae, Phoma spp. (including but not limited to Phoma sorghina,Phoma insidiosa, Phoma glumarum, Phoma glumicola and Phoma oryzina),Nigrospora spp. (including but not limited to Nigrospora oryzae,Nigrospora sphaerica, Nigrospora panici and Nigrospora padwickii),Epiococcum nigrum, Phyllostica spp., Wolkia decolorans, Monascuspurpureus, Aspergillus spp., Penicillium spp., Absidia spp., Mucor spp.,Chaetomium spp., Dematium spp., Monilia spp., Streptomyces spp.,Syncephalastrum spp., Veiticillium spp, Nematospora coryli, Nakatacasigmoidea, Cladosporium spp., Bipolaris spp., Coniothyrium spp.,Diplodia oryzae, Exserophilum rostratum, Helococera oryzae, Melanommaglumarum, Metashaeria spp., Mycosphaerella spp., Oidium spp., Pestalotiaspp., Phaeoseptoria spp., Sphaeropsis spp., Trematosphaerella spp., riceblack-streaked dwarf virus, rice dwarf virus, rice gall dwarf virus,barley yellow dwarf virus, rice grassy stunt virus, rice hoja blancavirus, rice necrosis mosaic virus, rice ragged stunt virus, rice stripevirus. rice stripe necrosis virus, rice transitory yellowing virus, ricetungro bacilliform virus, rice tungro spherical virus, rice yellowmottle virus, rice tarsonemid mite virus, Echinochloa hoja blanca virus,Echinochloa ragged stunt virus, orange leaf mycoplasma-like organism,yellow dwarf mycoplasma-like organism, Aphelenchoides besseyi,Ditylenchus angustus, Hirschmanniella spp., Criconemella spp.,Meloidogyne spp., Heterodera spp., Pratylenchus spp., Hoplolaimusindicus.

SOYBEANS: Phytophthora sojae, Fusarium solani f. sp. Glycines,Macrophomina phaseolina, Fusarium, Pythium, Rhizoctonia, Phialophoragregata, Sclerotinia sclerotionim, Diaporthe phaseolorum var. sojae,Colletotrichum truncatum, Phomopsis longicolla, Cercospora kikuchii,Diaporthe phaseolonum var. meridionalis (and var. caulivora), Phakopsorapachyrhyzi, Fusarium solani, Microsphaera diffusa, Septoria glycines,Cercospora kikuchii, Macrophomina phaseolina, Sclerotinia sclerotiorum,Corynespora cassiicola, Rhizoctonia solani, Cercospora sojina,Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Fusariumoxysporum, Diapothe phaseolorum var. sojae (Phomopsis sojae), Diaporthephaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii,Cercospora sojina, Peronospora manshurica, Colletotrichum dematium(Colletotichum truncatum), Corynespora cassiicola, Phyllostictasojicola, Alternaria alternata, Pseudomonas syringae p.v. glycinea,Xanthomonas campestris p.v. phaseoli, Microspaera diffusa, Fusariumsemitectum, Phialophora gregata, Soybean mosaic virus, Glomerellaglycines, Tobacco Ring spot virus, Tobacco Streak virus, Phakopsorapachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium dearyanum,Tomato spotted wilted virus, Heterodera glycines, Fusarium solani,Soybean cyst and root knot nematodes.

CORN: Fusarium moniliforme var. subglutinans, Erwinia stewartii,Fusarium moniliforme, Gibberella zeae (Fusarium Gramineanim),Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythiumdebaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum,Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T(cochliobolus heterostrophus), Helminthosporium carbonum I, II, and III(Cochliobolus carbonum), Exserohilum turcicum I, II and III,Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis,Kabatie-maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi,Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum,Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvuiariainaequalis, Curvularia pallescens, Clavibacter michiganese subsp.Nebraskense, Trichoderma viride, Maize dwarf Mosaic Virus A and B, WheatStreak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi,Pseudonomas avenae, Erwinia chrysantemi p.v. Zea, Erwinia corotovora,Cornstun spiroplasma, Diplodia macrospora, Sclerophthora macrospora,Peronosclerospora sorghi, Peronoscherospora philippinesis,Peronosclerospora maydis, Peronosclerospora sacchari, Spacelothecareiliana, Physopella zea, Cephalosporium maydis, Caphalosporiumacremonium, Maize Chlorotic Mottle Virus, High Plains Virus, MaizeMosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize StripeVirus, Maize Rought Dwarf Virus:

WHEAT: Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri,Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v.syringae, Alternaria alternata, Cladosporium herbarum, Fusariumgraminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici,Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola,Erysiphe graminis f. sp. Tritici, Puccinia graminis f. sp. Tritici,Puccinia recondite f. sp. tritici, puccinia striiformis, Pyrenophoratriticirepentis, Septoria nodorum, Septoria tritici, Spetoria avenae,Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctoniacerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum,Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, BarleyYellow Dwarf Virus, Brome Mosaic Virus, Soil Borne Wheat Mosaic Virus,Wheat Streak Virus, Wheat Spindle Streak Virus, American Wheat StriateVirus, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Pstilagotritici, Tilletia indica, Rhizoctonia solani, Pythium arrhenomannes,Pythium gramicola, Pythium aphanidermatum, High Plains Virus, EuropeanWheat Striate Virus:

CANOLA: Albugo candida, Alternaria brassicae, Leptosharia maculans,Rhizoctonia solani, Sclerotinia sclerotiorum, Mycospaerella brassiccola,Pythium ultimum, Peronospora parasitica, Fusarium roseum, Fusariumoxysporum, Tilletia foetida, Tilletia caries, Alternaria alternata:

SUNFLOWER: Plasmophora halstedii, Scherotinia sclerotiorum, AsterYellows, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi,Alternaria zinniae, Botrytis cinera, Phoma macdonaldii, Macrophominaphaseolina, Erysiphe cichoracearum, Phizopus oryzae, Rhizopus arrhizus,Rhizopus stolonifer, Puccinia helianthi, Verticillium Dahliae, Erwiniacarotovorum p.v. carotovora, Cephalosporium acremonium, Phytophthoracryptogea, Albugo tragopogonis.

SORGHUM: Exserohilum turcicum, Colletotrichum graminicola (Glomerellagraminicola), Cercospora sorghi, Gloeocercospora sorghi, Ascochytasorghi, Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v.holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrophominaphaseolina, Periconia circinata, Fusarium moniliforme, Alternariaalternate, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularialunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonasalboprecipitans), Ramulispora sorghi, Ramulispora sorghicola,Phyllachara sacchari Sporisorium relianum (Sphacelotheca reliana),Sphacelotheca cruenta, Sporisorium sorghi, Sugarcane mosaic H, MaizeDwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani,Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi,Peronosclerospora philippinensis, Sclerospora graminicola, Fusariumgraminearum, Fusarium Oxysporum, Pythium arrhenomanes, Pythiumgraminicola.

ALFALFA: Clavibater michiganensis subsp. Insidiosum, Pythium ultimum,Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythiumaphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phomamedicaginis var. medicaginis, Cercospora medicaginis, Pseudopezizamedicaginis, Leptotrochila medicaginis, Fusarium oxysporum, Rhizoctoniasolani, Uromyces striatus, Colletotrichum trifolii race 1 and race 2,Leptosphaerulina briosiana, Stemphylium botryosum, Stagonosporameliloti, Sclerotinia trifoliorum, Alfalfa Mosaic Virus, Verticilliumalbo-atrum, Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches,Stemphylium herbarum, Stemphylium alfalfae.

(v) Plant Agronomic Characteristics

Two of the factors determining where crop plants can be grown are theaverage daily temperature during the growing season and the length oftime between frosts. Within the areas where it is possible to grow aparticular crop, there are varying limitations on the maximal time it isallowed to grow to maturity and be harvested. For example, a variety tobe grown in a particular area is selected for its ability to mature anddry down to harvestable moisture content within the required period oftime with maximum possible yield. Therefore, crops of varying maturitiesare developed for different growing locations. Apart from the need todry down sufficiently to permit harvest, it is desirable to have maximaldrying take place in the field to minimize the amount of energy requiredfor additional drying post harvest. Also, the more readily a productsuch as grain can dry down, the more time there is available for growthand kernel fill. It is considered that genes that influence maturityand/or dry down can be identified and introduced into plant lines usingtransformation techniques to create new varieties adapted to differentgrowing locations or the same growing location, but having improvedyield to moisture ratio at harvest. Expression of genes that areinvolved in regulation of plant development may be especially useful.

It is contemplated that genes may be introduced into plants that wouldimprove standability and other plant growth characteristics. Expressionof novel genes in plants which confer stronger stalks, improved rootsystems, or prevent or reduce ear droppage or shattering would be ofgreat value to the farmer. It is proposed that introduction andexpression of genes that increase the total amount of photoassimilateavailable by, for example, increasing light distribution and/orinterception would be advantageous. In addition, the expression of genesthat increase the efficiency of photosynthesis and/or the leaf canopywould further increase gains in productivity. It is contemplated thatexpression of a phytochrome gene in crop plants may be advantageous.Expression of such a gene may reduce apical dominance, confersemidwarfism on a plant, or increase shade tolerance (U.S. Pat. No.5,268,526). Such approaches would allow for increased plant populationsin the field.

(vi) Nutrient Utilization

The ability to utilize available nutrients may be a limiting factor ingrowth of crop plants. It is proposed that it would be possible to alternutrient uptake, tolerate pH extremes, mobilization through the plant,storage pools, and availability for metabolic activities by theintroduction of novel genes. These modifications would allow a plant,for example, maize to more efficiently utilize available nutrients. Itis contemplated that an increase in the activity of, for example, anenzyme that is normally present in the plant and involved in nutrientutilization would increase the availability of a nutrient or decreasethe availability of an antinutritive factor. An example of such anenzyme would be phytase. It is further contemplated that enhancednitrogen utilization by a plant is desirable. Expression of a glutamatedehydrogenase gene in plants, e.g., E. coli gdhA genes, may lead toincreased fixation of nitrogen in organic compounds. Furthermore,expression of gdhA in plants may lead to enhanced resistance to theherbicide glufosinate by incorporation of excess ammonia into glutamate,thereby detoxifying the ammonia. It also is contemplated that expressionof a novel gene may make a nutrient source available that was previouslynot accessible, e.g., an enzyme that releases a component of nutrientvalue from a more complex molecule, perhaps a macromolecule.

Polypeptides useful for improving nitrogen flow, sensing, uptake,storage and/or transport include those involved in aspartate, glutamineor glutamate biosynthesis, polypeptides involved in aspartate, glutamineor glutamate transport, polypeptides associated with the TOR (Target ofRapamycin) pathway, nitrate transporters, nitrate reductases, aminotransferases, ammonium transporters, chlorate transporters orpolypeptides involved in tetrapyrrole biosynthesis.

Polypeptides useful for increasing the rate of photosynthesis includephytochrome, ribulose bisphosphate carboxylase-oxygenase, Rubiscoactivase, photosystem I and II proteins, electron carriers, ATPsynthase, NADH dehydrogenase or cytochrome oxidase.

Polypeptides useful for increasing phosphorus uptake, transport orutilization include phosphatases or phosphate transporters.

(vii) Male Sterility

Male sterility is useful in the production of hybrid seed. It isproposed that male sterility may be produced through expression of novelgenes. For example, it has been shown that expression of genes thatencode proteins, RNAs, or peptides that interfere with development ofthe male inflorescence and/or gametophyte result in male sterility.Chimeric ribonuclease genes that express in the anthers of transgenictobacco and oilseed rape have been demonstrated to lead to malesterility (Mariani et al., Nature, 347:737-741, 1990).

A number of mutations were discovered in maize that confer cytoplasmicmale sterility. One mutation in particular, referred to as T cytoplasm,also correlates with sensitivity to Southern corn leaf blight. A DNAsequence, designated TURF 13 (Levings, Science, 250:942-947, 1990), wasidentified that correlates with T cytoplasm. It is proposed that itwould be possible through the introduction of TURF 13 viatransformation, to separate male sterility from disease sensitivity. Asit is necessary to be able to restore male fertility for breedingpurposes and for grain production, it is proposed that genes encodingrestoration of male fertility also may be introduced.

(viii) Altered Nutritional Content

Genes may be introduced into plants to improve or alter the nutrientquality or content of a particular crop. Introduction of genes thatalter the nutrient composition of a crop may greatly enhance the feed orfood value. For example, the protein of many grains is suboptimal forfeed and food purposes, especially when fed to pigs, poultry, andhumans. The protein is deficient in several amino acids that areessential in the diet of these species, requiring the addition ofsupplements to the grain. Limiting essential amino acids may includelysine, methionine, tryptophan, threonine, valine, arginine, andhistidine. Some amino acids become limiting only after corn issupplemented with other inputs for feed formulations. The levels ofthese essential amino acids in seeds and grain may be elevated bymechanisms which include, but are not limited to, the introduction ofgenes to increase the biosynthesis of the amino acids, decrease thedegradation of the amino acids, increase the storage of the amino acidsin proteins, or increase transport of the amino acids to the seeds orgrain.

Polypeptides useful for providing increased seed protein quantity and/orquality include polypeptides involved in the metabolism of amino acidsin plants, particularly polypeptides involved in biosynthesis ofmethionine/cysteine and lysine, amino acid transporters, amino acidefflux carriers, seed storage proteins, proteases, or polypeptidesinvolved in phytic acid metabolism.

The protein composition of a crop may be altered to improve the balanceof amino acids in a variety of ways including elevating expression ofnative proteins, decreasing expression of those with poor composition,changing the composition of native proteins, or introducing genesencoding entirely new proteins possessing superior composition.

The introduction of genes that alter the oil content of a crop plant mayalso be of value. Increases in oil content may result in increases inmetabolizable-energy-content and density of the seeds for use in feedand food. The introduced genes may encode enzymes that remove or reducerate-limitations or regulated steps in fatty acid or lipid biosynthesis.Such genes may include, but are not limited to, those that encodeacetyl-CoA carboxylase, ACP-acyltransferase, alpha-ketoacyl-ACPsynthase, or other well known fatty acid biosynthetic activities. Otherpossibilities are genes that encode proteins that do not possessenzymatic activity such as acyl carrier protein. Genes may be introducedthat alter the balance of fatty acids present in the oil providing amore healthful or nutritive feedstuff. The introduced DNA also mayencode sequences that block expression of enzymes involved in fatty acidbiosynthesis, altering the proportions of fatty acids present in crops.

Genes may be introduced that enhance the nutritive value of crops, or offoods derived from crops by increasing the level of naturally occurringphytosterols, or by encoding for proteins to enable the synthesis ofphytosterols in crops. The phytosterols from these crops can beprocessed directly into foods, or extracted and used to manufacture foodproducts.

Genes may be introduced that enhance the nutritive value of the starchcomponent of crops, for example by increasing the degree of branching,resulting in improved utilization of the starch in livestock by delayingits metabolism. Additionally, other major constituents of a crop may bealtered, including genes that affect a variety of other nutritive,processing, or other quality aspects. For example, pigmentation may beincreased or decreased.

Carbohydrate metabolism may be altered, for example by increased sucroseproduction and/or transport. Polypeptides useful for affecting oncarbohydrate metabolism include polypeptides involved in sucrose orstarch metabolism, carbon assimilation or carbohydrate transport,including, for example sucrose transporters or glucose/hexosetransporters, enzymes involved in glycolysis/gluconeogenesis, thepentose phosphate cycle, or raffinose biosynthesis, or polypeptidesinvolved in glucose signaling, such as SNF1 complex proteins.

Feed or food crops may also possess sub-optimal quantities of vitamins,antioxidants or other nutraceuticals, requiring supplementation toprovide adequate nutritive value and ideal health value. Introduction ofgenes that enhance vitamin biosynthesis may be envisioned including, forexample, vitamins A, E, B12, choline, or the like. Mineral content mayalso be sub-optimal. Thus genes that affect the accumulation oravailability of compounds containing phosphorus, sulfur, calcium,manganese, zinc, or iron among others would be valuable.

Numerous other examples of improvements of crops may be used with theinvention. The improvements may not necessarily involve grain, but may,for example, improve the value of a crop for silage. Introduction of DNAto accomplish this might include sequences that alter lignin productionsuch as those that result in the “brown midrib” phenotype associatedwith superior feed value for cattle. Other genes may encode for enzymesthat alter the structure of extracellular carbohydrates in the stover,or that facilitate the degradation of the carbohydrates in the non-grainportion of the crop so that it can be efficiently fermented into ethanolor other useful carbohydrates.

It may be desirable to modify the nutritional content of plants byreducing undesirable components such as fats, starches, etc. This may bedone, for example, by the use of exogenous nucleic acids that encodeenzymes which increase plant use or metabolism of such components sothat they are present at lower quantities. Alternatively, it may be doneby use of exogenous nucleic acids that reduce expression levels oractivity of native plant enzymes that synthesize such components.

Likewise the elimination of certain undesirable traits may improve thefood or feed value of the crop. Many undesirable traits must currentlybe eliminated by special post-harvest processing steps and the degree towhich these can be engineered into the plant prior to harvest andprocessing would provide significant value. Examples of such traits arethe elimination of anti-nutritionals such as phytates and phenoliccompounds which are commonly found in many crop species. Also, thereduction of fats, carbohydrates and certain phytohormones may bevaluable for the food and feed industries as they may allow a moreefficient mechanism to meet specific dietary requirements.

In addition to direct improvements in feed or food value, genes also maybe introduced which improve the processing of crops and improve thevalue of the products resulting from the processing. One use of crops isvia wetmilling. Thus novel genes that increase the efficiency and reducethe cost of such processing, for example by decreasing steeping time,may also find use. Improving the value of wetmilling products mayinclude altering the quantity or quality of starch, oil, corn glutenmeal, or the components of gluten feed. Elevation of starch may beachieved through the identification and elimination of rate limitingsteps in starch biosynthesis by expressing increased amounts of enzymesinvolved in biosynthesis or by decreasing levels of the other componentsof crops resulting in proportional increases in starch.

Oil is another product of wetmilling, the value of which may be improvedby introduction and expression of genes. Oil properties may be alteredto improve its performance in the production and use of cooking oil,shortenings, lubricants or other oil-derived products or improvement ofits health attributes when used in the food-related applications. Novelfatty acids also may be synthesized which upon extraction can serve asstarting materials for chemical syntheses. The changes in oil propertiesmay be achieved by altering the type, level, or lipid arrangement of thefatty acids present in the oil. This in turn may be accomplished by theaddition of genes that encode enzymes that catalyze the synthesis ofnovel fatty acids (e.g. fatty acid elongases, desaturases) and thelipids possessing them or by increasing levels of native fatty acidswhile possibly reducing levels of precursors or breakdown products.Alternatively, DNA sequences may be introduced which slow or block stepsin fatty acid biosynthesis resulting in the increase in precursor fattyacid intermediates. Genes that might be added include desaturases,epoxidases, hydratases, dehydratases, or other enzymes that catalyzereactions involving fatty acid intermediates. Representative examples ofcatalytic steps that might be blocked include the desaturations fromstearic to oleic acid or oleic to linolenic acid resulting in therespective accumulations of stearic and oleic acids. Another example isthe blockage of elongation steps resulting in the accumulation of C8 toC12 saturated fatty acids.

Polypeptides useful for providing increased seed oil quantity and/orquality include polypeptides involved in fatty acid and glycerolipidbiosynthesis, beta-oxidation enzymes, enzymes involved in biosynthesisof nutritional compounds, such as carotenoids and tocopherols, orpolypeptides that increase embryo size or number or thickness ofaleurone.

Polypeptides involved in production of galactomannans orarabinogalactans are of interest for providing plants having increasedand/or modified reserve polysaccharides for use in food, pharmaceutical,cosmetic, paper and paint industries.

Polypeptides involved in modification of flavonoid/isoflavonoidmetabolism in plants include cinnamate-4-hydroxylase, chalcone synthaseor flavones synthase. Enhanced or reduced activity of such polypeptidesin modified plants will provide changes in the quantity and/or speed offlavonoid metabolism in plants and may improve disease resistance byenhancing synthesis of protective secondary metabolites or improvingsignaling pathways governing disease resistance.

Polypeptides involved in lignin biosynthesis are of interest forincreasing plants' resistance to lodging and for increasing theusefulness of plant materials as befouls.

(ix) Production or Assimilation of Chemicals or Biological

It may further be considered that a modified plant prepared inaccordance with the invention may be used for the production ormanufacturing of useful biological compounds that were either notproduced at all, or not produced at the same level, in the corn plantpreviously. Alternatively, plants produced in accordance with theinvention may be made to metabolize or absorb and concentrate certaincompounds, such as hazardous wastes, thereby allowing bioremediation ofthese compounds.

The novel plants producing these compounds are made possible by theintroduction and expression of one or potentially many genes with theconstructs provided by the invention. The vast array of possibilitiesinclude but are not limited to any biological compound which ispresently produced by any organism such as proteins, nucleic acids,primary and intermediary metabolites, carbohydrate polymers, enzymes foruses in bioremediation, enzymes for modifying pathways that producesecondary plant metabolites such as falconoid or vitamins, enzymes thatcould produce pharmaceuticals, and for introducing enzymes that couldproduce compounds of interest to the manufacturing industry such asspecialty chemicals and plastics. The compounds may be produced by theplant, extracted upon harvest and/or processing, and used for anypresently recognized useful purpose such as pharmaceuticals, fragrances,and industrial enzymes to name a few.

(x) Other Characteristics

Cell cycle modification: Polypeptides encoding cell cycle enzymes andregulators of the cell cycle pathway are useful for manipulating growthrate in plants to provide early vigor and accelerated maturation.Improvements in quality traits, such as seed oil content, may also beobtained by expression of cell cycle enzymes and cell cycle regulators.Polypeptides of interest for modification of cell cycle pathway includecycling and EIF5alpha pathway proteins, polypeptides involved inpolyamine metabolism, polypeptides which act as regulators of the cellcycle pathway, including cyclin-dependent kinases (CDKs), CDK-activatingkinases, cell cycle-dependent phosphatases, CDK-inhibitors, Rb andRb-binding proteins, or transcription factors that activate genesinvolved in cell proliferation and division, such as the E2F family oftranscription factors, proteins involved in degradation of cyclins, suchas cullins, and plant homologs of tumor suppressor polypeptides.

Plant growth regulators: Polypeptides involved in production ofsubstances that regulate the growth of various plant tissues are ofinterest in the present invention and may be used to provide modifiedplants having altered morphologies and improved plant growth anddevelopment profiles leading to improvements in yield and stressresponse. Of particular interest are polypeptides involved in thebiosynthesis, or degradation of plant growth hormones, such asgibberellins, brassinosteroids, cytokinins, auxins, ethylene or abscisicacid, and other proteins involved in the activity, uptake and/ortransport of such polypeptides, including for example, cytokininoxidase, cytokinin/purine permeases, F-box proteins, G-proteins orphytosulfokines.

Transcription factors in plants: Transcription factors play a key rolein plant growth and development by controlling the expression of one ormore genes in temporal, spatial and physiological specific patterns.Enhanced or reduced activity of such polypeptides in modified plantswill provide significant changes in gene transcription patterns andprovide a variety of beneficial effects in plant growth, development andresponse to environmental conditions. Transcription factors of interestinclude, but are not limited to myb transcription factors, includinghelix-turn-helix proteins, homeodomain transcription factors, leucinezipper transcription factors, MADS transcription factors, transcriptionfactors having AP2 domains, zinc finger transcription factors, CCAATbinding transcription factors, ethylene responsive transcriptionfactors, transcription initiation factors or UV damaged DNA bindingproteins.

Homologous recombination: Increasing the rate of homologousrecombination in plants is useful for accelerating the introgression oftransgenes into breeding varieties by backcrossing, and to enhance theconventional breeding process by allowing rare recombinants betweenclosely linked genes in phase repulsion to be identified more easily.Polypeptides useful for expression in plants to provide increasedhomologous recombination include polypeptides involved in mitosis and/ormeiosis, DNA replication, nucleic acid metabolism, DNA repair pathwaysor homologous recombination pathways including for example,recombinases, nucleases, proteins binding to DNA double-strand breaks,single-strand DNA binding proteins, strand-exchange proteins,resolvases, ligases, helicases and polypeptide members of the RAD52epistasis group.

Non-Protein-Expressing Exogenous Nucleic Acids

Plants with decreased expression of a gene of interest can also beachieved, for example, by expression of antisense nucleic acids, dsRNAor RNAi, catalytic RNA such as ribozymes, sense expression constructsthat exhibit cosuppression effects, aptamers or zinc finger proteins.

Antisense RNA reduces production of the polypeptide product of thetarget messenger RNA, for example by blocking translation throughformation of RNA:RNA duplexes or by inducing degradation of the targetmRNA. Antisense approaches are a way of preventing or reducing genefunction by targeting the genetic material as disclosed in U.S. Pat.Nos. 4,801,540; 5,107,065; 5,759,829; 5,910,444; 6,184,439; and6,198,026, all of which are. incorporated herein by reference. In oneapproach, an antisense gene sequence is introduced that is transcribedinto antisense RNA that is complementary to the target mRNA. Forexample, part or all of the normal gene sequences are placed under apromoter in inverted orientation so that the wrong or complementarystrand is transcribed into a non-protein expressing antisense RNA. Thepromoter used for the antisense gene may influence the level, timing,tissue, specificity, or inducibility of the antisense inhibition.

Autonomous mini-chromosomes may contain exogenous DNA bounded byrecombination sites, for example lox-P sites, that can be recognized bya recombinase, e.g. Cre, and removed from the mini-chromosome. In caseswhere there is a homologous recombination site or sites in the hostgenomic DNA, the exogenous DNA excised the mini-chromosome may beintegrated into the genome at one of the specific recombination sitesand the DNA bounded by the recombination sites will become integratedinto the host DNA. The use of a mini-chromosome as a platform for DNAexcision or for launching such DNA integration into the host genome mayinclude in vivo induction of the expression of a recombinase encoded inthe genomic DNA of a transgenic host, or in a mini-chromosome or otherepisome.

RNAi gene suppression in plants by transcription of a dsRNA is describedin U.S. Pat. No. 6,506,559, U.S. patent application Publication No.2002/0168707, WO 98/53083, WO 99/53050 and WO 99/61631, all of which areincorporated herein by reference. The double-stranded RNA or RNAiconstructs can trigger the sequence-specific degradation of the targetmessenger RNA. Suppression of a gene by RNAi can be achieved using arecombinant DNA construct having a promoter operably linked to a DNAelement comprising a sense and anti-sense element of a segment ofgenomic DNA of the gene, e.g., a segment of at least about 23nucleotides, more preferably about 50 to 200 nucleotides where the senseand anti-sense DNA components can be directly linked or joined by anintron or artificial DNA segment that can form a loop when thetranscribed RNA hybridizes to form a hairpin structure.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of the target gene or genes or facilitate molecularreactions. Ribozymes are targeted to a given sequence by hybridizationof sequences within the ribozyme to the target mRNA. Two stretches ofhomology are required for this targeting, and these stretches ofhomologous sequences flank the catalytic ribozyme structure. It ispossible to design ribozymes that specifically pair with virtually anytarget mRNA and cleave the target mRNA at a specific location, therebyinactivating it. A number of classes of ribozymes have been identified.One class of ribozymes is derived from a number of small circular RNAsthat are capable of self-cleavage and replication in plants. The RNAsreplicate either alone (viroid RNAs) or with a helper virus (satelliteRNAs). Examples include Tobacco Ringspot Virus (Prody et al., Science,231:1577-1580, 1986), Avocado Sunblotch Viroid (Palukaitis et al.,Virology, 99:145-151, 1979; Symons, Nucl. Acids Res., 9:6527-6537,1981), and Lucerne Transient Streak Virus (Forster and Symons, Cell,49:211-220, 1987), and the satellite RNAs from velvet tobacco mottlevirus, Solanum nodiflorum mottle virus and subterranean clover mottlevirus. The design and use of target RNA-specific ribozymes is describedin Haseloff, et al., Nature 334:585-591 (1988). Several differentribozyme motifs have been described with RNA cleavage activity (Symons,Annu. Rev. Biocheni., 61:641-671, 1992). Other suitable ribozymesinclude sequences from RNase P with RNA cleavage activity (Yuan et al.,Proc. Natl. Acad. Sci. USA, 89:8006-8010, 1992; Yuan and Altman,Science, 263:1269-1273, 1994; U.S. Pat. Nos. 5,168,053 and 5,624,824),hairpin ribozyme structures (Berzal-Herranz et al., Genes and Devel.,6:129-134, 1992; Chowrira et al., J. Biol. Chem., 269:25856-25864, 1994)and Hepatitis Delta virus based ribozymes (U.S. Pat. No. 5,625,047). Thegeneral design and optimization of ribozyme directed RNA cleavageactivity has been discussed in detail (Haseloff and Gerlach, 1988,Nature. Aug. 18, 1988;334(6183):585-91, Chowrira et al., J. Biol. Chem.,269:25856-25864, 1994).

Another method of reducing protein expression utilizes the phenomenon ofcosuppression or gene silencing (for example, U.S. Pat. Nos. 6,063,947;5,686,649; or 5,283,184; each of which is incorporated herein byreference). Cosuppression of an endogenous gene using a full-length cDNAsequence as well as a partial cDNA sequence are known (for example,Napoli et al., Plant Cell 2:279-289 [1990]; van der Krol et al, PlantCell 2:291-299 [1990]; Smith et al., Mol. Gen. Genetics 224:477-481[1990]). The phenomenon of cosuppression has also been used to inhibitplant target genes in a tissue-specific manner.

In some embodiments, nucleic acids from one species of plant areexpressed in another species of plant to effect cosuppression of ahomologous gene. The introduced sequence generally will be substantiallyidentical to the endogenous sequence intended to be repressed, forexample, about 65%, 80%, 85%, 90%, or preferably 95% or greateridentical. Higher identity may result in a more effective repression ofexpression of the endogenous sequence. A higher identity in a shorterthan full length sequence compensates for a longer, less identicalsequence. Furthermore, the introduced sequence need not have the sameintron or exon pattern, and identity of non-coding segments will beequally effective. Generally, where inhibition of expression is desired,some transcription of the introduced sequence occurs. The effect mayoccur where the introduced sequence contains no coding sequence per se,but only intron or untranslated sequences homologous to sequencespresent in the primary transcript of the endogenous sequence.

Yet another method of reducing protein activity is by expressing nucleicacid ligands, so-called aptamers, which specifically bind to theprotein. Aptamers may be obtained by the SELEX (Systematic Evolution ofLigands by EXponential Enrichment) method. See U.S. Pat. No. 5,270,163,incorporated herein by reference. In the SELEX method, a candidatemixture of single stranded nucleic acids having regions of randomizedsequence is contacted with the protein and those nucleic acids having anincreased affinity to the target are selected and amplified. Afterseveral iterations a nucleic acid with optimal affinity to thepolypeptide is obtained and is used for expression in modified plants.

A zinc finger protein that binds a polypeptide-encoding sequence or itsregulatory region is also used to alter expression of the nucleotidesequence. Transcription of the nucleotide sequence may be reduced orincreased. Zinc finger proteins are, for example, described in Beerli etal. (1998) PNAS 95:14628-14633., or in WO 95/19431, WO 98/54311, or WO96/06166, all incorporated herein by reference.

Other examples of non-protein expressing sequences specificallyenvisioned for use with the invention include tRNA sequences, forexample, to alter codon usage, and rRNA variants, for example, which mayconfer resistance to various agents such as antibiotics.

It is contemplated that unexpressed DNA sequences, including novelsynthetic sequences, could be introduced into cells as proprietary“labels” of those cells and plants and seeds thereof. It would not benecessary for a label DNA element to disrupt the function of a geneendogenous to the host organism, as the sole function of this DNA wouldbe to identify the origin of the organism. For example, one couldintroduce a unique DNA sequence into a plant and this DNA element wouldidentify all cells, plants, and progeny of these cells as having arisenfrom that labeled source. It is proposed that inclusion of label DNAswould enable one to distinguish proprietary germplasm or germplasmderived from such, from unlabelled germplasm.

Exemplary Plant Promoters, Regulatory Sequences and Targeting Sequences

Exemplary classes of plant promoters are described below.

Constitutive Expression promoters: Exemplary constitutive expressionpromoters include the ubiquitin promoter (e.g., sunflower—Binet et al.Plant Science 79: 87-94 (1991); maize—Christensen et al. Plant Molec.Biol. 12: 619-632 (1989); and Arabidopsis—Callis et al., J. Biol. Chem.265: 12486-12493 (1990) and Norris et al., Plant Mol. Biol. 21: 895-906(1993)); the CaMV 35S promoter (U.S. Pat. Nos. 5,858,742 and 5,322,938);or the actin promoter (e.g., rice—U.S. Pat. No. 5,641,876; McElroy etal. Plant Cell 2: 163-171 (1990), McElroy et al. Mol. Gen. Genet. 231:150-160 (1991), and Chibbar et al. Plant Cell Rep. 12: 506-509 (1993)).

Inducible Expression promoters: Exemplary inducible expression promotersinclude the chemically regulatable tobacco PR-1 promoter (e.g.,tobacco—U.S. Pat. No. 5,614,395; Arabidopsis—Lebel et al., Plant J. 16:223-233 (1998); maize—U.S. Pat. No. 6,429,362). Various chemicalregulators may be employed to induce expression, including thebenzothiadiazole, isonicotinic acid, and salicylic acid compoundsdisclosed in U.S. Pat. Nos. 5,523,311 and 5,614,395. Other promotersinducible by certain alcohols or ketones, such as ethanol, include, forexample, the alcA gene promoter from Aspergillus nidulans (Caddick etal. (1998) Nat. Biotechnol 16:177-180). A glucocorticoid-mediatedinduction system is described in Aoyama and Chua (1997) The PlantJournal 11: 605-612 wherein gene expression is induced by application ofa glucocorticoid, for example a dexamethasone. Another class of usefulpromoters are water-deficit-inducible promoters, e.g. promoters whichare derived from the 5′ regulatory region of genes identified as a heatshock protein 17.5 gene (HSP 17.5), an HVA22 gene (HVA22), and acinnamic acid 4-hydroxylase (CA4H) gene (CA4H) of Zea maize. Anotherwater-deficit-inducible promoter is derived from the rab-17 promoter asdisclosed by Vilardell et al., Plant Molecular Biology, 17(5):985-993,1990. See also U.S. Pat. No. 6,084,089 which discloses cold induciblepromoters, U.S. Pat. No. 6,294,714 which discloses light induciblepromoters, U.S. Pat. No. 6,140,078 which discloses salt induciblepromoters, U.S. Pat. No. 6,252,138 which discloses pathogen induciblepromoters, and U.S. Pat. No. 6,175,060 which discloses phosphorusdeficiency inducible promoters.

As another example, numerous wound-inducible promoters have beendescribed (e.g. Xu et al. Plant Molec. Biol. 22: 573-588 (1993),Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, PlantMolec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22:129-142 (1993), Warner et al. Plant J. 3: 191-201 (1993)). Logemanndescribe 5′ upstream sequences of the potato wunl gene. Xu et al. showthat a wound-inducible promoter from the dicotyledon potato (pin2) isactive in the monocotyledon rice. Rohrmeier & Lehle describe maize WiplcDNA which is wound induced and which can be used to isolate the cognatepromoter. Firek et al. and Warner et al. have described a wound-inducedgene from the monocotyledon Asparagus officinalis, which is expressed atlocal wound and pathogen invasion sites.

Tissue-Specific Promoters: Exemplary promoters that express genes onlyin certain tissues are useful according to the present invention. Forexample root specific expression may be attained using the promoter ofthe maize metallothionein-like (MTL) gene described by de Framond (FEBS290: 103-106 (1991)) and also in U.S. Pat. No. 5,466,785, incorporatedherein by reference. U.S. Pat. No. 5,837,848 discloses a root specificpromoter. Another exemplary promoter confers pith-preferred expression(see Int'l. Pub. No. WO 93/07278, herein incorporated by reference,which describes the maize trpA gene and promoter that is preferentiallyexpressed in pith cells). Leaf-specific expression may be attained, forexample, by using the promoter for a maize gene encoding phosphoenolcarboxylase (PEPC) (see Hudspeth & Grula, Plant Molec Biol 12: 579-589(1989)). Pollen-specific expression may be conferred by the promoter forthe maize calcium-dependent protein kinase (CDPK) gene which isexpressed in pollen cells (WO 93/07278). U.S. Pat. Appl. Pub. No.20040016025 describes tissue-specific promoters. Pollen-specificexpression may be conferred by the tomato LAT52 pollen-specific promoter(Bate et. al., Plan mol Biol. 1998 July;37(5):859-69).

See also U.S. Pat. No. 6,437,217 which discloses a root-specific maizeRS81 promoter, U.S. Pat. No. 6,426,446 which discloses a root specificmaize RS324 promoter, U.S. Pat. No. 6,232,526 which discloses aconstitutive maize A3 promoter, U.S. Pat. No. 6,177,611 which disclosesconstitutive maize promoters, U.S. Pat. No. 6,433,252 which discloses amaize L3 oleosin promoter that are aleurone and seed coat-specificpromoters, U.S. Pat. No. 6,429,357 which discloses a constitutive riceactin 2 promoter and intron, U.S. patent application Pub. No.20040216189 which discloses an inducible constitutive leaf specificmaize chloroplast aldolase promoter.

Optionally a plant transcriptional terminator can be used in place ofthe plant-expressed gene native transcriptional terminator. Exemplarytranscriptional terminators are those that are known to function inplants and include the CaMV 35S terminator, the tml terminator, thenopaline synthase terminator and the pea rbcS E9 terminator. These canbe used in both monocotyledons and dicotyledons.

Various intron sequences have been shown to enhance expression,particularly in monocotyledonous cells. For example, the introns of themaize Adhl gene have been found to significantly enhance expression.Intron 1 was found to be particularly effective and enhanced expressionin fusion constructs with the chloramphenicol acetyltransferase gene(Callis et al., Genes Develop. 1: 1183-1200 (1987)). The intron from themaize bronzel gene also enhances expression. Intron sequences have beenroutinely incorporated into plant transformation vectors, typicallywithin the non-translated leader. U.S. Patent Application Publication2002/0192813 discloses 5′, 3′ and intron elements useful in the designof effective plant expression vectors.

A number of non-translated leader sequences derived from viruses arealso known to enhance expression, and these are particularly effectivein dicotyledonous cells. Specifically, leader sequences from TobaccoMosaic Virus (TMV, the “omega-sequence”), Maize Chlorotic Mottle Virus(MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effectivein enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15:8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)).Other leader sequences known in the art include but are not limited to:picomavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′noncoding region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. PNAS USA86:6126-6130 (1989)); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al., 1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20); human immunoglobulin heavy-chainbinding protein (BiP) leader, (Macejak, D. G., and Samow, P., Nature353: 90-94 (1991); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L.,Nature 325:622-625 (1987); tobacco mosaic virus leader (TMV), (Gallie etal., Molecular Biology of RNA, pages 237-256 (1989); or Maize ChloroticMottle Virus leader (MCMV) (Lommel et al., Virology 81:382-385 (1991).See also, Della-Cioppa et al., Plant Physiology 84:965-968 (1987).

A minimal promoter may also be incorporated. Such a promoter has lowbackground activity in plants when there is no transactivator present orwhen enhancer or response element binding sites are absent. Oneexemplary minimal promoter is the Bz1 minimal promoter, which isobtained from the bronzel gene of maize. Roth et al., Plant Cell 3: 317(1991). A minimal promoter may also be created by use of a syntheticTATA element. The TATA element allows recognition of the promoter by RNApolymerase factors and confers a basal level of gene expression in theabsence of activation (see generally, Mukumoto (1993) Plant Mol Biol 23:995-1003; Green (2000) Trends Biochem Sci 25: 59-63).

Sequences controlling the targeting of gene products also may beincluded. For example, the targeting of gene products to the chloroplastis controlled by a signal sequence found at the amino terminal end ofvarious proteins which is cleaved during chloroplast import to yield themature protein (e.g. Comai et al. J. Biol. Chem. 263: 15104-15109(1988)). These signal sequences can be fused to heterologous geneproducts to effect the import of heterologous products into thechloroplast (van den Broeck, et al. Nature 313: 358-363 (1985)). DNAencoding for appropriate signal sequences can be isolated from the 5′end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSPsynthase enzyme, the GS2 protein or many other proteins which are knownto be chloroplast localized. Other gene products are localized to otherorganelles such as the mitochondrion and the peroxisome (e.g. Unger etal. Plant Molec. Biol. 13: 411-418 (1989)). Examples of sequences thattarget to such organelles are the nuclear-encoded ATPases or specificaspartate amino transferase isoforms for mitochondria. Targetingcellular protein bodies has been described by Rogers et al. (Proc. Natl.Acad. Sci. USA 82: 6512-6516 (1985)). In addition, amino terminal andcarboxy-terminal sequences are responsible for targeting to the ER, theapoplast, and extracellular secretion from alcurone cells (Koehler & Ho,Plant Cell 2: 769-783 (1990)). Additionally, amino terminal sequences inconjunction with carboxy terminal sequences are responsible for vacuolartargeting of gene products (Shinshi et al. Plant Molec. Biol. 14:357-368 (1990)).

Another possible element which may be introduced is a matrix attachmentregion element (MAR), such as the chicken lysozyme A element (Stief,1989), which can be positioned around an expressible gene of interest toeffect an increase in overall expression of the gene and diminishposition dependent effects upon incorporation into the plant genome(Stief et al., Nature, 341:343, 1989; Phi-Van et al., Mol. Cell. Biol.,10:2302-2307.1990).

Use of Non-Plant Promoter Regions Isolated from Drosophila Melanogasterand Saccharomyces Cerevisiae to Express Genes in Plants

The promoter in the mini-chromosome of the present invention can bederived from plant or non-plant species. In a preferred embodiment, thenucleotide sequence of the promoter is derived from non-plant speciesfor the expression of genes in plant cells, including but not limited todicotyledon plant cells such as tobacco, tomato, potato, soybean,canola, sunflower, alfalfa, cotton and Arabidopsis, or monocotyledonousplant cell, such as wheat, maize, rye, rice, turf grass, oat, barley,sorghum, millet, and sugarcane. In one embodiment, the non-plantpromoters are constitutive or inducible promoters derived from insect,e.g., Drosophila melanogaster or yeast, e.g., Saccharomyces cerevisiae.Table 2 lists the promoters from Drosophila melanogaster andSaccharomyces cerevisiae that are used to derive the examples ofnon-plant promoters in the present invention. Promoters derived from anyanimal, protist, or fungi are also contemplated. SEQ ID NOS: 4-23 areexamples of promoter sequences derived from Drosophila melanogaster orSaccharomyces cerevisiae. These non-plant promoters can be operablylinked to nucleic acid sequences encoding polypeptides ornon-protein-expressing sequences including, but not limited to,antisense RNA and ribozymes, to form nucleic acid constructs, vectors,and host cells (prokaryotic or eukaryotic), comprising the promoters.TABLE 2 Drosophila melanogaster Promoters (Information obtained from theFlybase Web Site at http://flybase.bio.indiana.edu/which is a databaseof the Drosophila Genome) Standard promoter gene Seq Id No. SymbolFlybase ID name Gene Product Chromosome  4 gd FBgn0004654Phosphogluconate 6- X dehydrogenase phosphogluconate dehydrogenase  5rim FBgn0015946 grim grim-P138 3  5 ro FBgn0003961 Urate oxidase Uro-P12  7 na FBgn0003448 snail sna-P1 2  8 h3 FBgn0003249 Rhodopsin 3 Rh3 3 9 sp-1 γ FBgn0002564 Larval serum Lsp1γ-P1 3 protein 1 γ Saccharomycescerevisiae Promoters (Information obtained from the Saccharomyces GenomeDatabase Web site at http://www.yeastgenome.org/SearchContents.shtmlStandard Systematic promoter gene Seq No. Symbol Name name Gene ProductChromosome 10 ef-2 YBR118W TEF2 Translation 2 (Translation elongationfactor elongation factor EF-1 alpha promtoer) 11 eu-1 YGL009C LEU1(LEUcine isopropylmalate 7 biosynthesis) isomerase 12 et16 YPR167CMEThionine 3′phosphoadenyly 16 requiring lsulfate reductase 13 eu-2YCL018W LEU2 (leucine beta-IPM 3 biosynthesis) (isopropylmalate)dehydrogenase 14 is-4 YCL030C HIS4 (HIStidine histidinol 3 requiring)dehydrogenase 15 et-2 YNL277W MET2 L-homoserine-O- 14 (methionineacetyltransferase requiring) 16 te-3 YKL178C STE3 (alias a-factorreceptor 11 DAF2 Sterile) 17 rg-1 YOL058W ARG1 (alias arginosuccinate 15ARG10 synthetase ARGinine requiring) 18 gk-1 YCR012W PGK1phosphoglycerate 3 (phosphoglycerate kinase kinase) 19 PD-1 YDL022W GPD1(alias glycerol-3- 4 DAR1/HOR1/OSG1/ phosphate OSR5: dehydrogenaseglycerol-3- phosphate dehydrogenase activity 20 DH1 YOL086C ADH1 (aliasalcohol 15 ADC1) dehydrogenase 21 PD-2 YOL059W GPD2 (alias glycerol-3-15 GPD3: glycerol- phosphate 3-phosphate dehydrogenase dehydrogenaseactivity 22 rg-4 YHR018C ARGinine argininosuccinate 8 requiring lyase 23at-1 YAR035W YAT-1 (carnitine carnitine 1 acetyltransferase)acetyltransferase

The present invention relates to methods for producing a polypeptide,comprising cultivating plant material for the production of thepolypeptide at any level, wherein the plant host cells comprises a firstnucleic acid sequence encoding the polypeptide operably linked to asecond nucleic acid sequence comprising a heterologous promoter foreignto the nucleic acid sequence, wherein the promoter comprises a sequenceselected from the group consisting of SEQ ID NOS:4 to 23 or subsequencesthereof; and mutant, hybrid, or tandem promoters thereof that retainpromoter activity.

The present invention also relates to methods for producing non-proteinexpressed sequences, comprising cultivating plant material for theproduction of the non-protein expressed sequence, wherein the plant hostcell comprises a first nucleic acid sequence encoding the non-proteinexpressed sequences operably linked to a second nucleic acid sequencecomprising a heterologous promoter foreign to the nucleic acid sequence,wherein the promoter comprises a sequence selected from the groupconsisting of SEQ ID NOS:4 to 23 or subsequences thereof; and mutant,hybrid, or tandem promoters thereof.

The present invention also relates to isolated promoter sequences and toconstructs, vectors, or plant host cells comprising one or more of thepromoters operably linked to a nucleic acid sequence encoding apolypeptide or non-protein expressing sequence.

In the methods of the present invention, the promoter may also be amutant of the promoters having a substitution, deletion, and/orinsertion of one or more nucleotides in the nucleic acid sequence of SEQID NOS:4 to 23.

The present invention also relates to methods for obtaining derivativepromoters of SEQ ID NOS:4 to 23.

The techniques used to isolate or clone a nucleic acid sequencecomprising a promoter of interest are known in the art and includeisolation from genomic DNA. The cloning procedures may involve excisionor amplification, for example by polymerase chain reaction, andisolation of a desired nucleic acid fragment comprising the nucleic acidsequence encoding the promoter, insertion of the fragment into a vectormolecule, and incorporation of the recombinant vector into the plantcell.

Definitions

The term “adchromosomal” plant or plant part as used herein means aplant or plant part that contains functional, stable and autonomousmini-chromosomes. Adchromosomal plants or plant parts may be chimeric ornot chimeric (chimeric meaning that mini-chromosomes are only in certainportions of the plant, and are not uniformly distributed throughout theplant). An adchromosomal plant cell contains at least one functional,stable and autonomous mini-chromosome.

The term “autonomous” as used herein means that when delivered to plantcells, at least some mini-chromosomes are transmitted through mitoticdivision to daughter cells and are episomal in the daughter plant cells,i.e. are not chromosomally integrated in the daughter plant cells.Daughter plant cells that contain autonomous mini-chromosomes can beselected for further replication using, for example, selectable orscreenable markers. During the introduction into a cell of amini-chromosome, or during subsequent stages of the cell cycle, theremay be chromosomal integration of some portion or all of the DNA derivedfrom a mini-chromosome in some cells. The mini-chromosome is stillcharacterized as autonomous despite the occurrence of such events if aplant may be regenerated that contains episomal descendants of themini-chromosome distributed throughout its parts, or if gametes orprogeny can be derived from the plant that contain episomal descendantsof the mini-chromosome distributed through its parts.

As used herein, a “centromere” is any DNA sequence that confers anability to segregate to daughter cells through cell division. In onecontext, this sequence may produce a transmission efficiency to daughtercells ranging from about 1% to about 100%, including to about 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 95% of daughter cells.Variations in such a transmission efficiency may find importantapplications within the scope of the invention; for example,mili-chromosomes carrying centromeres that confer 100% stability couldbe maintained in all daughter cells without selection, while those thatconfer 1% stability could be temporarily introduced into a transgenicorganism, but be eliminated when desired. In particular embodiments ofthe invention, the centromere may confer stable transmission to daughtercells of a nucleic acid sequence, including a recombinant constructcomprising the centromere, through mitotic or meiotic divisions,including through both meiotic and meiotic divisions. A plant centromereis not necessarily derived from plants, but has the ability to promoteDNA transmission to daughter plant cells.

As used herein, the term “circular permutations” refer to variants of asequence that begin at base n within the sequence, proceed to the end ofthe sequence, resume with base number one of the sequence, and proceedto base n−1. For this analysis, n may be any number less than or equalto the length of the sequence. For example, circular permutations of thesequence ABCD are: ABCD, BCDA, CDAB, and DABC.

The term “co-delivery” as used herein refers to the delivery of twonucleic acid segments to a cell. In co-delivery of plant growth inducinggenes and mini-chromosomes, the two nucleic acid segments are deliveredsimultaneously using the same delivery method. Alternatively, thenucleic acid segment containing the growth inducing gene, optionally aspart of an episomal vector, such as a viral vector or a plasmid vector,may be delivered to the plant cells before or after delivery of themini-chromosome, and the mini-chromosome may carry an exogenous nucleicacid that induces expression of the earlier-delivered growth inducinggene. In this embodiment, the two nucleic acid segments may be deliveredseparately at different times provided the encoded growth inducingfactors are functional during the appropriate time period.

The term “coding sequence” is defined herein as a nucleic acid sequencethat is transcribed into mRNA which is translated into a polypeptidewhen placed under the control of promoter sequences. The boundaries ofthe coding sequence are generally determined by the ATG start codonlocated at the start of the open reading frame, near the 5′ end of themRNA, and TAG, TGA or TAA stop codons at the end of the coding sequence,near the 3′ end f the mRNA, and in some cases, a transcriptionterminator sequence located just downstream of the open reading frame atthe 3′ end of the mRNA. A coding sequence can include, but is notlimited to, genomic DNA, cDNA, semisynthetic, synthetic, or recombinantnucleic acid sequences.

As used herein the term “consensus” refers to a nucleic acid sequencederived by comparing two or more related sequences. A consensus sequencedefines both the conserved and variable sites between the sequencesbeing compared. Any one of the sequences used to derive the consensus orany permutation defined by the consensus may be useful in constructionof mini-chromosomes.

The term “exogenous” when used in reference to a nucleic acid, forexample, is intended to refer to any nucleic acid that has beenintroduced into a recipient cell, regardless of whether the same orsimilar nucleic acid is already present in such a cell. Thus, as anexample, “exogenous DNA” can include an additional copy of DNA that isalready present in the plant cell, DNA from another plant, DNA from adifferent organism, or a DNA generated externally, such as a DNAsequence containing an antisense message of a gene, or a DNA sequenceencoding a synthetic or modified version of a gene. An “exogenous gene”can be a gene not normally found in the host genome in an identicalcontext, or an extra copy of a host gene. The gene may be isolated froma different species than that of the host genome, or alternatively,isolated from the host genome but operably linked to one or moreregulatory regions which differ from those found in the unaltered,native gene.

The term “functional” as used herein to describe a mini-chromosome meansthat when an exogenous nucleic acid is present within themini-chromosome the exogenous nucleic acid can function in a detectablemanner when the mini-chromosome is within a plant cell; exemplaryfunctions of the exogenous nucleic acid include transcription of theexogenous nucleic acid, expression of the exogenous nucleic acid,regulatory control of expression of other exogenous nucleic acids,recognition by a restriction enzyme or other endonuclease, ribozyme orrecombinase; providing a substrate for DNA methylation, DNA glycolationor other DNA chemical modification; binding to proteins such ashistones, helix-loop-helix proteins, zinc binding proteins, leucinezipper proteins, MADS box proteins, topoisomerases, helicases,transposases, TATA box binding proteins, viral protein, reversetranscriptases, or cohesins; providing an integration site forhomologous recombination; providing an integration site for atransposon, T-DNA or retrovirus; providing a substrate for RNAisynthesis; priming of DNA replication; aptamer binding; or kinetochorebinding. If multiple exogenous nucleic acids are present within themini-chromosome, the function of one or preferably more of the exogenousnucleic acids can be detected under suitable conditions permittingfunction thereof.

As used herein, a “library” is a pool of cloned DNA fragments thatrepresents some or all DNA sequences collected, prepared or purifiedfrom a specific source. Each library may contain the DNA of a givenorganism inserted as discrete restriction enzyme generated fragments oras randomly sheared fragments into many thousands of plasmid vectors.For purposes of the present invention, E. coli, yeast, and Salmonellaplasmids are particularly useful for propagating the genome inserts fromother organisms. In principle, any gene or sequence present in thestarting DNA preparation can be isolated by screening the library with aspecific hybridization probe (see, for example, Young et al., In:Eukaryotic Genetic Systems ICN-UCLA Symposia on Molecular and CellularBiology, VII, 315-331, 1977).

As used herein, the term “linker” refers to a DNA molecule, generally upto 50 or 60 nucleotides long and composed of two or more complementaryoligonucleotides that have been synthesized chemically, or excised oramplified from existing plasmids or vectors. In a preferred embodiment,this fragment contains one, or preferably more than one, restrictionenzyme site for a blunt cutting enzyme and/or a staggered cuttingenzyme, such as BamHI. One end of the linker is designed to be ligatableto one end of a linear DNA molecule and the other end is designed to beligatable to the other end of the linear molecule, or both ends may bedesigned to be ligatable to both ends of the linear DNA molecule.

As used herein, a “mini-chromosome” is a recombinant DNA constructincluding a centromere and capable of transmission to daughter cells. Amini-chromosome may remain separate from the host genome (as episomes)or may integrate into host chromosomes. The stability of this constructthrough cell division could range between from about 1% to about 100%,including about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% andabout 95%. The mini-chromosome construct may be a circular or linearmolecule. It may include elements such as one or more telomeres, originof replication sequences, stuffer sequences, buffer sequences, chromatinpackaging sequences, linkers and genes. The number of such sequencesincluded is only limited by the physical size limitations of theconstruct itself. It could contain DNA derived from a naturalcentromere, although it may be preferable to limit the amount of DNA tothe minimal amount required to obtain a transmission efficiency in therange of 1-100%. The mini-chromosome could also contain a syntheticcentromere composed of tandem arrays of repeats of any sequence, eitherderived from a natural centromere, or of synthetic DNA. Themini-chromosome could also contain DNA derived from multiple naturalcentromeres. The mini-chromosome may be inherited through mitosis ormeiosis, or through both meiosis and mitosis. As used herein, the termmini-chromosome specifically encompasses and includes the terms “plantartificial chromosome” or “PLAC,” or engineered chromosomes ormicrochromosomes and all teachings relevant to a PLAC or plantartificial chromosome specifically apply to constructs within themeaning of the term mini-chromosome.

The term “non-protein expressing sequence” or “non-protein codingsequence” is defined herein as a nucleic acid sequence that is noteventually translated into protein. The nucleic acid may or may not betranscribed into RNA. Exemplary sequences include ribozymes or antisenseRNA.

The term “operably linked” is defined herein as a configuration in whicha control sequence, e.g., a promoter sequence, directs transcription ortranslation of another sequence, for example a coding sequence. Forexample, a promoter sequence could be appropriately placed at a positionrelative to a coding sequence such that the control sequence directs theproduction of a polypeptide encoded by the coding sequence.

“Phenotype” or “phenotypic trait(s)”, as used herein, refers to anobservable property or set of properties resulting from the expressionof a gene. The set of properties may be observed visually or afterbiological or biochemical testing, and may be constantly present or mayonly manifest upon challenge with the appropriate stimulus or activationwith the appropriate signal.

The term “plant,” as used herein, refers to any type of plant. Exemplarytypes of plants are listed below, but other types of plants will beknown to those of skill in the art and could be used with the invention.Modified plants of the invention include, for example, dicots,gymnosperm, monocots, mosses, ferns, horsetails, club mosses, liverworts, hornworts, red algae, brown algae, gametophytes and sporophytesof pteridophytes, and green algae.

The term “crop plant” refers to plants grown for agricultural orcommercial rather than experimental purposes and specifically excludesArabidopsis thaliana. Some plants grown for experimental purposes maytake on commercial importance when used to produce pharmaceutical orchemical products. Centromeres “derived from crop plants” according tothe present invention specifically exclude centromeres that arefragments of naturally occurring Arabidopsis thaliana centromeres ornaturally occurring descendants thereof. Centromeres derived from cropplants include variants (mutants) of Arabidopsis thaliana centromeres,or artificial centromeres synthesized based on nucleotide sequences ofArabidopsis thaliana centromeres.

A common class of plants exploited in agriculture are vegetable crops,including artichokes, kohlrabi, arugula, leeks, asparagus, lettuce(e.g., head, leaf, romaine), bok choy, malanga, broccoli, melons (e.g.,muskmelon, watermelon, crenshaw, honeydew, cantaloupe), brusselssprouts, cabbage, cardoni, carrots, napa, cauliflower, okra, onions,celery, parsley, chick peas, parsnips, chicory, chinese cabbage,peppers, collards, potatoes, cucumber plants (marrows, cucumbers),pumpkins, cucurbits, radishes, dry bulb onions, rutabaga, eggplant,salsify, escarole, shallots, endive, garlic, spinach, green onions,squash, greens, beet (sugar beet or fodder beet), sweet potatoes, swisschard, horseradish, tomatoes, kale, turnips, or spices.

Other types of plants frequently finding commercial use include fruitand vine crops such as apples, grapes, apricots, cherries, nectarines,peaches, pears, plums, prunes, quince, almonds, chestnuts, filberts,pecans, pistachios, walnuts, citrus, blueberries, boysenberries,cranberries, currants, loganberries, raspberries, strawberries,blackberries, grapes, avocados, bananas, kiwi, persimmons, pomegranate,pineapple, tropical fruits, pomes, melon, mango, papaya, or lychee.

Modified wood and fiber or pulp plants of particular interest include,but are not limited to maple, oak, cherry, mahogany, poplar, aspen,birch, beech, spruce, fir, kenaf, pine, walnut, cedar, redwood,chestnut, acacia, bombax, alder, eucalyptus, catalpa, mulberry,persimmon, ash, honeylocust, sweetgum, privet, sycamore, magnolia,sourwood, cottonwood, mesquite, buckthorn, locust, willow, elderberry,teak, linden, bubinga, basswood or elm.

Modified flowers and ornamental plants of particular interest, include,but are not limited to, roses, petunias, pansy, peony, olive, begonias,violets, phlox, nasturtiums, irises, lilies, orchids, vinca,philodendron, poinsettias, opuntia, cyclamen, magnolia, dogwood, azalea,redbud, boxwood, Viburnum, maple, elderberry, hosta, agave, asters,sunflower, pansies, hibiscus, morning glory, alstromeria, zinnia,geranium, Prosopis, artemesia, clematis, delphinium, dianthus, gallium,coreopsis, iberis, lamium, poppy, lavender, leucophyllum, sedum, salvia,verbascum, digitalis, penstemon, savory, pythrethrum, or oenothera.Modified nut-bearing trees of particular interest include, but are notlimited to pecans, walnuts, macadamia nuts, hazelnuts, almonds, orpistachios, cashews, pignolas or chestnuts.

Many of the most widely grown plants are field crop plants such asevening primrose, meadow foam, corn (field, sweet, popcorn), hops,jojoba, peanuts, rice, safflower, small grains (barley, oats, rye,wheat, etc.), sorghum, tobacco, kapok, leguminous plants (beans,lentils, peas, soybeans), oil plants (rape, mustard, poppy, olives,sunflowers, coconut, castor oil plants, cocoa beans, groundnuts, oilpalms), fibre plants (cotton, flax, hemp, jute), lauraceae (cinnamon,camphor), or plants such as coffee, sugarcane, cocoa, tea, or naturalrubber plants. Still other examples of plants include bedding plantssuch as flowers, cactus, succulents or ornamental plants, as well astrees such as forest (broad-leaved trees or evergreens, such asconifers), fruit, ornamental, or nut-bearing trees, as well as shrubs orother nursery stock.

Still other examples of plants include bedding plants such as flowers,cactus, succulents or ornamental plants, as well as trees such as forest(broad-leaved trees or evergreens, such as conifers), fruit, ornamental,or nut-bearing trees, as well as shrubs or other nursery stock.

Modified crop plants of particular interest in the present inventioninclude, but are not limited to, soybean (including the variety known asGlycine max), cotton, canola (also known as rape), wheat, sunflower,sorghum, alfalfa, barley, safflower, millet, rice, tobacco, fruit andvegetable crops or turfgrasses. Exemplary cereals include maize, wheat,barley, oats, rye, millet, sorghum, rice triticale, secale, einkorn,spelt, emmer, teff, milo, flax, gramma grass, Tripsacum sp., orteosinte. Oil-producing plants include plant species that produce andstore triacylglycerol in specific organs, primarily in seeds. Suchspecies include soybean (Glycine max), rapeseed or canola (includingBrassica napus, Brassica rapa or Brassica campestris), Brassica juncea,Brassica carinata, sunflower (Helianthus annus), cotton (Gossypiumhirsutum), corn (Zea mays), cocoa (Theobroma cacao), safflower(Carthamus tinctorius), oil palm (Elaeis guineensis), coconut palm(Cocos nucifera), flax (Linum usitatissimum), castor (Ricinus communis)or peanut (Arachis hypogaea).

The term “plant part” as used herein includes pollen, silk, endosperm,ovule, seed, embryo, pods, roots, cuttings, tubers, stems, stalks,fruit, berries, nuts, flowers, leaves, bark, wood, whole plant, plantcell, plant organ, protoplast, cell culture, crown, callus culture,petiole, petal, sepal, stamen, stigma, style, bud, or any group of plantcells organized into a structural and functional unit. In one preferredembodiment, the exogenous nucleic acid is expressed in a specificlocation or tissue of a plant, for example, epidermis, vascular tissue,meristem, cambium, cortex, pith, leaf, sheath, flower, root or seed.

The term “promoter” is defined herein as a DNA sequence that allows thebinding of RNA polymerase (including but not limited to RNA polymeraseI, RNA polymerase II and RNA polymerase III from eukaryotes) and directsthe polymerase to a downstream transcriptional start site of a nucleicacid sequence encoding a polypeptide to initiate transcription. RNApolymerase effectively catalyzes the assembly of messenger RNAcomplementary to the appropriate DNA strand of the coding region.

A “promoter operably linked to a heterologous gene” is a promoter thatis operably linked to a gene that is different from the gene to whichthe promoter is normally operably linked in its native state. Similarly,an “exogenous nucleic acid operably linked to a heterologous regulatorysequence” is a nucleic acid that is operably linked to a regulatorycontrol sequence to which it is not normally linked in its native state.

The term “hybrid promoter” is defined herein as parts of two or morepromoters that are fused together to generate a sequence that is afusion of the two or more promoters, which is operably linked to acoding sequence and mediates the transcription of the coding sequenceinto mRNA.

The term “tandem promoter” is defined herein as two or more promotersequences each of which is operably linked to a coding sequence andmediates the transcription of the coding sequence into mRNA.

The term “constitutive active promoter” is defined herein as a promoterthat allows permanent stable expression of the gene of interest.

The term “Inducible promoter” is defined herein as a promoter induced bythe presence or absence of biotic or an abiotic factor.

The term “polypeptide” does not refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “exogenous polypeptide” is defined as a polypeptidewhich is not native to the plant cell, a native polypeptide in whichmodifications have been made to alter the native sequence, or a nativepolypeptide whose expression is quantitatively altered as a result of amanipulation of the plant cell by recombinant DNA techniques.

As used herein, the term “pseudogene” refers to a non-functional copy ofa protein-coding gene; pseudogenes found in the genomes of eukaryoticorganisms are often inactivated by mutations and are thus presumed to benon-essential to that organism; pseudogenes of reverse transcriptase andother open reading frames found in retroelements are abundant in thecentromeric regions of Arabidopsis and other organisms and are oftenpresent in complex clusters of related sequences.

As used herein the term “regulatory sequence” refers to any DNA sequencethat influences the efficiency of transcription or translation of anygene. The term includes, but is not limited to, sequences comprisingpromoters, enhancers and terminators.

As used herein the term “repeated nucleotide sequence” refers to anynucleic acid sequence of at least 25 bp present in a genome or arecombinant molecule, other than a telomere repeat, that occurs at leasttwo or more times and that are preferably at least 80% identical eitherin head to tail or head to head orientation either with or withoutintervening sequence between repeat units.

As used herein, the term “retroelement” or “retrotransposon” refers to agenetic element related to retroviruses that disperse through an RNAstage; the abundant retroelements present in plant genomes contain longterminal repeats (LTR retrotransposons) and encode a polyprotein genethat is processed into several proteins including a reversetranscriptase. Specific retroelements (complete or partial sequences)can be found in and around plant centromeres and can be present asdispersed copies or complex repeat clusters. Individual copies ofretroelements may be truncated or contain mutations; intact retrolementsare rarely encountered.

As used herein the term “satellite DNA” refers to short DNA sequences(typically <1000 bp) present in a genome as multiple repeats, mostlyarranged in a tandemly repeated fashion, as opposed to a dispersedfashion. Repetitive arrays of specific satellite repeats are abundant inthe centromeres of many higher eukaryotic organisms.

As used herein, a “screenable marker” is a gene whose presence resultsin an identifiable phenotype. This phenotype may be observable understandard conditions, altered conditions such as elevated temperature, orin the presence of certain chemicals used to detect the phenotype. Theuse of a screenable marker allows for the use of lower, sub-killingantibiotic concentrations and the use of a visible marker gene toidentify clusters of transformed cells, and then manipulation of thesecells to homogeneity. Preferred screenable markers of the presentinclude genes that encode fluorescent proteins that are detectable by avisual microscope such as the fluorescent reporter genes DsRed, ZsGreen,ZsYellow, AmCyan, Green Fluorescent Protein (GFP). An additionalpreferred screenable marker gene is lac.

The invention also contemplates novel methods of screening foradchromosomal plant cells that involve use of relatively low,sub-killing concentrations of selection agent (e.g. sub-killingantibiotic concentrations), and also involve use of a screenable marker(e.g., a visible marker gene) to identify clusters of modified cellscarrying the screenable marker, after which these screenable cells aremanipulated to homogeneity. As used herein, a “selectable marker” is agene whose presence results in a clear phenotype, and most often agrowth advantage for cells that contain the marker. This growthadvantage may be present under standard conditions, altered conditionssuch as elevated temperature, specialized media compositions, or in thepresence of certain chemicals such as herbicides or antibiotics. Use ofselectable markers is described, for example, in Broach et al. Gene,8:121-133, 1979. Examples of selectable markers include the thymidinekinase gene, the cellular adenine phosphoribosyltransferase gene and thedihydrylfolate reductase gene, hygromycin phosphotransferase genes, thebar gene, neomycin phosphotransferase genes and phosphomannoseisomerase, among others. Preferred selectable markers in the presentinvention include genes whose expression confer antibiotic or herbicideresistance to the host cell, or proteins allowing utilization of acarbon source not normally utilized by plant cells. Expression of one ofthese markers should be sufficient to enable the maintenance of a vectorwithin the host cell, and facilitate the manipulation of the plasmidinto new host cells. Of particular interest in the present invention areproteins conferring cellular resistance to kanamycin, G 418,paramomycin, hygromycin, bialaphos, and glyphosate for example, orproteins allowing utilization of a carbon source, such as mannose, notnormally utilized by plant cells.

The term “stable” as used herein means that the mini-chromosome can betransmitted to daughter cells over at least 8 mitotic generations. Someembodiments of mini-chromosomes may be transmitted as functional,autonomous units for less than 8 mitotic generations, e.g. 1, 2, 3, 4,5, 6, or 7. Preferred mini-chromosomes can be transmitted over at least8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 or 30 generations, for example, through theregeneration or differentiation of an entire plant, and preferably aretransmitted through meiotic division to gametes. Other preferredmini-chromosomes can be further maintained in the zygote derived fromsuch a gamete or in an embryo or endosperm derived from one or more suchgametes. A “functional and stable” mini-chromosome is one in whichfunctional mini-chromosomes can be detected after transmission of themini-chromosomes over at least 8 mitotic generations, or afterinheritance through a meiotic division. During mitotic division, asoccurs occasionally with native chromosomes, there may be somenon-transmission of mini-chromosomes; the mini-chromosome may still becharacterized as stable despite the occurrence of such events if anadchromosomal plant that contains descendants of the mini-chromosomedistributed throughout its parts may be regenerated from cells,cuttings, propagules, or cell cultures containing the mini-chromosome,or if an adchromosomal plant can be identified in progeny of the plantcontaining the mini-chromosome.

As used herein, a “structural gene” is a sequence which codes for apolypeptide or RNA and includes 5′ and 3′ ends. The structural gene maybe from the host into which the structural gene is transformed or fromanother species. A structural gene will preferably, but not necessarily,include one or more regulatory sequences which modulate the expressionof the structural gene, such as a promoter, terminator or enhancer. Astructural gene will preferably, but not necessarily, confer some usefulphenotype upon an organism comprising the structural gene, for example,herbicide resistance. In one embodiment of the invention, a structuralgene may encode an RNA sequence which is not translated into a protein,for example a tRNA or rRNA gene.

As used herein, the term “telomere” or “telomere DNA” refers to asequence capable of capping the ends of a chromosome, thereby preventingdegradation of the chromosome end, ensuring replication and preventingfusion to other chromosome sequences. Telomeres can include naturallyoccurring telomere sequences or synthetic sequences. Telomeres from onespecies may confer telomere activity in another species. An exemplarytelomere DNA is a heptanucleotide telomere repeat TTTAGGG (and itscomplement) found in the majority of plants.

“Transformed,” “transgenic,” “modified,” and “recombinant” refer to ahost organism such as a plant into which an exogenous or heterologousnucleic acid molecule has been introduced, and includes meiocytes,seeds, zygotes, embryos, endosperm, or progeny of such plant that retainthe exogenous or heterologous nucleic acid molecule but which have notthemselves been subjected to the transformation process.

When the phrase “transmission efficiency” of a certain percent is used,transmission percent efficiency is calculated by measuringmini-chromosome presence through one or more mitotic or meioticgenerations. It is directly measured as the ratio (expressed as apercentage) of the daughter cells or plants demonstrating presence ofthe mini-chromosome to parental cells or plants demonstrating presenceof the mini-chromosome. Presence of the mini-chromosome in parental anddaughter cells is demonstrated with assays that detect the presence ofan exogenous nucleic acid carried on the mini-chromosome. Exemplaryassays can be the detection of a screenable marker (e.g. presence of afluorescent protein or any gene whose expression results in anobservable phenotype), a selectable marker, or PCR amplification of anyexogenous nucleic acid carried on the mini-chromosome.

Constructing Mini-Chromosomes by Site-Specific Recombination

Plant mini-chromosomes may be constructed using site-specificrecombination sequences (for example those recognized by thebacteriophage P1 Cre recombinase, or the bacteriophage lambda integrase,or similar recombination enzymes). A compatible recombination site, or apair of such sites, is present on both the centromere containing DNAclones and the donor DNA clones. Incubation of the donor clone and thecentromere clone in the presence of the recombinase enzyme causes strandexchange to occur between the recombination sites in the two plasmids;the resulting mini-chromosomes contain centromere sequences as well asmini-chromosome vector sequences. The DNA molecules formed in suchrecombination reactions is introduced into E. coli, other bacteria,yeast or plant cells by common methods in the field including, but notlimited to, heat shock, chemical transformation, electroporation,particle bombardment, whiskers, or other transformation method followedby selection for marker genes including chemical, enzymatic, color, orother marker present on either parental plasmid, allowing for theselection of transformants harboring mini-chromosomes.

II. Methods of Detecting and Characterizing Mini-Chromosomes in PlantCells or of Scoring Mini-Chromosome Performance in Plant Cells:

Identification of Candidate Centromere Fragments by Probing BACLibraries

Centromere clones are identified from a large genomic insert librarysuch as a Bacterial Artificial Chromosome library. Probes are labeledusing nick-translation in the presence of radioactively labeled dCTP,dATP, dGTP or dTTP as in, for example, the commercially availableRediprime kit (Amersham) as per the manufacturer's instructions. Otherlabeling methods familiar to those skilled in the art could besubstituted. The libraries are screened and deconvoluted. Genomic clonesare screened by probing with small centromere-specific clones (forexample 14F1 was used) which shows high homology to the satellitesequence (14F1 showed homology to “BJCANRD”, Genbank ID X68786.1). Otherembodiments of this procedure would involve hybridizing a library withother centromere sequences. Of the BAC clones identified using thisprocedure, a representative set are identified as having highhybridization signals to some probes, and optionally low hybridizationsignals to other probes. These are selected, the bacterial clones grownup in cultures and DNA prepared by methods familiar to those skilled inthe art such as alkaline lysis. The DNA composition of purified clonesare surveyed using for example fingerprinting by digesting withrestriction enzymes such as, but not limited to, HinfI or HindIII. In apreferred embodiment the restriction enzyme cuts within the tandemcentromere satellite repeat (see below). A variety of clones showingdifferent fingerprints are selected for conversion into mini-chromosomesand inheritance testing. It can also be informative to use multiplerestriction enzymes for fingerprinting or other enzymes which can cleaveDNA.

Fingerprinting Analysis of BACs and Mini-Chromosomes

Centromere function may be associated with large tandem arrays ofsatellite repeats. To assess the composition and architecture of thecentromere BACs, the candidate BACs are digested with a restrictionenzyme, such as HindIII, which cuts with known frequency within theconsensus sequence of the unit repeat of the tandemly repeatedcentromere satellite. Digestion products are then separated by agarosegel electrophoresis. Large insert clones containing a large array oftandem repeats will produce a strong band of the unit repeat size, aswell as less intense bands at 2× and 3× the unit repeat size, andfurther multiples of the repeat size. These methods are well-known andthere are many possible variations known to those skilled in the art.

Determining Sequence Composition of Mini-Chromosomes by ShotgunCloning/Sequencing, Sequence Analysis

To determine the sequence composition of the mini-chromosome, the insertis sequenced. To generate DNA suitable for sequencing mini-chromosomesare fragmented, for example by using a random shearing method (such assonication, nebulization, etc). Other fragmentation techniques may alsobe used such as enzymatic digestion. These fragments are then clonedinto a plasmid vector and sequenced. The resulting DNA sequence istrimmed of poor-quality sequence and of sequence corresponding to theplasmid vector. The sequence is then compared to the known DNA sequencesusing an algorithm such as BLAST to search a sequence database such asGenBank.

To determine the consensus of the satellite repeat in themini-chromosome, the sequences containing satellite repeat are alignedusing a DNA sequence alignment program such as ContigExpress from VectorNTI. The sequences may also be aligned to previously determined repeatsfor that species. The sequences are trimmed to unit repeat length usingthe consensus as a template. Sequences trimmed from the ends of thealignment are realigned with the consensus and further trimmed until allsequences are at or below the consensus length. The sequences are thenaligned with each other. The consensus is determined by the frequency ofa specific nucleotide at each position; if the most frequent base isthree times more frequent than the next most frequent base, it wasconsidered the consensus.

Methods for determining consensus sequence are well known in the art,see, e.g., U.S. Pat. App. Pub. No. 20030124561; Hall & Preuss (2002).These methods, including DNA sequencing, assembly, and analysis, arewell-known and there are many possible variations known to those skilledin the art. Other alignment parameters may also be useful such as usingmore or less stringent definitions of consensus.

Non-Selective Mini-Chromosome Mitotic Inheritance Assays

The following list of assays and potential outcomes illustrates howvarious assays can be used to distinguish autonomous events fromintegrated events.

Assay #1: Transient Assay

Mini-chromosomes are tested for their ability to become established aschromosomes and their ability to be inherited in mitotic cell divisions.In this assay, mini-chromosomes are delivered to plant cells, forexample Brassica suspension cells in liquid culture. The cells used canbe at various stages of growth. In this example, a population in whichsome cells were undergoing division was used. The mini-chromosome isthen assessed over the course of several cell divisions, by tracking thepresence of a screenable marker, e.g. a visible marker gene such as afluorescent protein. Mini-chromosomes that are inherited well may showan initial delivery into many single cells; after several celldivisions, these single cells divide to form clusters ofmini-chromosome-containing cells. Other exemplary embodiments of thismethod include delivering mini-chromosomes to other mitotic cell types,including roots and shoot meristems.

Assay #2: Non-Lineage Based Inheritance Assays on Modified TransformedCells and Plants

Mini-chromosome inheritance is assessed on modified cell lines andplants by following the presence of the mini-chromosome over the courseof multiple cell divisions. An initial population of mini-chromosomecontaining cells is assayed for the presence of the mini-chromosome, bythe presence of a marker gene, including but not limited to afluorescent protein, a colored protein, a protein assayable byhistochemical assay, and a gene affecting cell morphology. All nucleiare stained with a DNA-specific dye including but not limited to DAPI,Hoechst 33258, OliGreen, Giemsa YOYO, or TOTO, allowing a determinationof the number of cells that do not contain the mini-chromosome. Afterthe initial determination of the percent of cells carrying themini-chromosome, the cells are allowed to divide over the course ofseveral cell divisions. The number of cell divisions, n, is determinedby a method including but not limited to monitoring the change in totalweight of cells, and monitoring the change in volume of the cells or bydirectly counting cells in an aliquot of the culture. After a number ofcell divisions, the population of cells is again assayed for thepresence of the mini-chromosome. The loss rate per generation iscalculated by the equation:Loss rate per generation=1−(F/I)^(1/n)

The population of mini-chromosome-containing cells may includesuspension cells, roots, leaves, meristems, flowers, or any other tissueof modified plants, or any other cell type containing a mini-chromosome.

These methods are well-known and there are many possible variationsknown to those skilled in the art; they have been used before with humancells and yeast cells.

Assay #3: Lineage Based Inheritance Assays on Modified Cells and Plants

Mini-chromosome inheritance is assessed on modified cell lines andplants by following the presence of the mini-chromosome over the courseof multiple cell divisions. In cell types that allow for tracking ofcell lineage, including but not limited to root cell files, trichomes,and leaf stomata guard cells, mini-chromosome loss per generation doesnot need to be determined statistically over a population, it can bediscerned directly through successive cell divisions. In othermanifestations of this method, cell lineage can be discerned from cellposition, or methods including but not limited to the use ofhistological lineage tracing dyes, and the induction of genetic mosaicsin dividing cells.

In one simple example, the two guard cells of the stomata are daughtersof a single precursor cell. To assay mini-chromosome inheritance in thiscell type, the epidermis of the leaf of a plant containing amini-chromosome is examined for the presence of the mini-chromosome bythe presence of a marker gene, including but not limited to afluorescent protein, a colored protein, a protein assayable byhistochemical assay, and a gene affecting cell morphology. The number ofloss events in which one guard cell contains the mini-chromosome (L) andthe number of cell divisions in which both guard cells contain themini-chromosome (B) are counted. The loss rate per cell division isdetermined as L/(L+B). Other lineage-based cell types are assayed insimilar fashion. These methods are well-known and there are manypossible variations known to those skilled in the art; they have beenused before with yeast cells.

Lineal mini-chromosome inheritance may also be assessed by examiningroot files (e.g. Brassica root files) or clustered cells in callus (e.g.soybean callus) over time. Changes in the percent of cells carrying themini-chromosome will indicate the mitotic inheritance.

Assay #4: Inheritance Assays on Modified Cells and Plants in thePresence of Chromosome Loss Agents

Any of the above three assays can be done in the presence of chromosomeloss agents (including but not limited to coichicine, colcemid,caffeine, etopocide, nocodazole, oryzalin, trifluran). It is likely thatan autonomous mini-chromosome will prove more susceptible to lossinduced by chromosome loss agents; therefore, autonomousmini-chromosomes should show a lower rate of inheritance in the presenceof chromosome loss agents. These methods have been used to studychromosome loss in fruit flies and yeast; there are many possiblevariations known to those skilled in the art.

III. Transformation of Plant Cells and Plant Regeneration

Various methods may be used to deliver DNA into plant cells. Theseinclude biological methods, such as Agrobacterium, E. coli, and viruses,physical methods such as biolistic particle bombardment, nanocopoieadevice, the Stein beam gun silicon, carbide whiskers and microinjection,electrical methods such as electroporation, and chemical methods such asthe use of poly-ethylene glycol and other compounds known to stimulateDNA uptake into cells. Examples of these techniques are described byPaszkowski et al., EMBO J 3: 2717-2722 (1984), Potrykus et al., Mol.Gen. Genet. 199: 169-177 (1985), Reich et al., Biotechnology 4:1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987).Transformation using silicon carbide whiskers, e.g. in maize, isdescribed in Brisibe, J. Exp. Bot. 51(343):187-196 (2000) and Dunwell,Methods Mol. Biol. 111:375-82 (1999) and U.S. Pat. No. 5,464,765.

Agrobacterium-Mediated Delivery

Agrobacterium-mediated transformation is one method for introducing adesired genetic element into a plant. Several Agrobacterium speciesmediate the transfer of a specific DNA known as “T-DNA” that can begenetically engineered to carry a desired piece of DNA into many plantspecies. Plasmids used for delivery contain the T-DNA flanking thenucleic acid to be inserted into the plant. The major events marking theprocess of T-DNA mediated pathogenesis are induction of virulence genes,processing and transfer of T-DNA.

There are three common methods to transform plant cells withAgrobacterium. The first method is co-cultivation of Agrobacterium withcultured isolated protoplasts. This method requires an establishedculture system that allows culturing protoplasts and plant regenerationfrom cultured protoplasts. The second method is transformation of cellsor tissues with Agrobacterium. This method requires (a) that the plantcells or tissues can be modified by Agrobacterium and (b) that themodified cells or tissues can be induced to regenerate into wholeplants. The third method is transformation of seeds, apices or meristemswith Agrobacterium. This method requires exposure of the meristematiccells of these tissues to Agrobacterium and micropropagation of theshoots or plan organs arising from these meristematic cells.

Those of skill in the art are familiar with procedures for growth andsuitable culture conditions for Agrobacterium as well as subsequentinoculation procedures. Liquid or semi-solid culture media can be used.The density of the Agrobacterium culture used for inoculation and theratio of Agrobacterium cells to explant can vary from one system to thenext, as can media, growth procedures, timing and lighting conditions.

Tranformation of dicotyledons using Agrobacterium has long been known inthe art, and transformation of monocotyledons using Agrobacterium hasalso been described. See, WO 94/00977 and U.S. Pat. No. 5,591,616, bothof which are incorporated herein by reference. See also, Negrotto etal., Plant Cell Reports 19: 798-803 (2000), incorporated herein byreference.

A number of wild-type and disarmed strains of Agrobacterium tumefaciensand Agrobacterium rhizogenes harboring Ti or Ri plasmids can be used forgene transfer into plants. Preferably, the Agrobacterium hosts containdisarmed Ti and Ri plasmids that do not contain the oncogenes that causetumorigenesis or rhizogenesis. Exemplary strains include Agrobacteriumtumefaciens strain C58, a nopaline-type strain that is used to mediatethe transfer of DNA into a plant cell, octopine-type strains such asLBA4404 or succinamopine-type strains, e.g., EHA101 or EHA105. The useof these strains for plant transformation has been reported and themethods are familiar to those of skill in the art.

U.S. Application No. 20040244075 published Dec. 2, 2004 describesimproved methods of Agrobacterium-mediated transformation. Theefficiency of transformation by Agrobacterium may be enhanced by using anumber of methods known in the art. For example, the inclusion of anatural wound response molecule such as acetosyringone (AS) to theAgrobacterium culture has been shown to enhance transformationefficiency with Agrobacterium tumefaciens (Shahla et al., (1987) PlantMolec. Biol. 8:291-298). Alternatively, transformation efficiency may beenhanced by wounding the target tissue to be modified or transformed.Wounding of plant tissue may be achieved, for example, by punching,maceration, bombardment with microprojectiles, etc. (See e.g., Bidney etal., (1992) Plant Molec. Biol. 18:301-313).

In addition, a recent method described by Broothaerts, et. al. (Nature433: 629-633, 2005) expands the bacterial genera that can be used totransfer genes into plants. This work involved the transfer of adisarmed Ti plasmid without T-DNA and another vector with T-DNAcontaining the marker enzyme beta-glucuronidase, into three differentbacteria. Gene transfer was successful and this method significantlyexpands the tools available for gene delivery into plants.

Microprojectile Bombardment Delivery

Another widely used technique to genetically transform plants involvesthe use of microprojectile bombardment. In this process, a nucleic acidcontaining the desired genetic elements to be introduced into the plantis deposited on or in small dense particles, e.g., tungsten, platinum,or preferably 1 micron gold particles, which are then delivered at ahigh velocity into the plant tissue or plant cells using a specializedbiolistics device. Many such devices have been designed and constructed;one in particular, the PDS1000/He sold by BioRad, is the instrument mostcommonly used for biolistics of plant cells. The advantage of thismethod is that no specialized sequences need to be present on thenucleic acid molecule to be delivered into plant cells; delivery of anynucleic acid sequence is theoretically possible.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos, seedlingexplants, or any plant tissue or target cells may be arranged on solidculture medium. The cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate.

Various biolistics protocols have been described that differ in the typeof particle or the manner in which DNA is coated onto the particle. Anytechnique for coating microprojectiles that allows for delivery oftransforming DNA to the target cells may be used. For example, particlesmay be prepared by functionalizing the surface of a gold oxide particle,providing free amine groups. DNA, having a strong negative charge, bindsto the functionalized particles.

Parameters such as the concentration of DNA used to coatmicroprojectiles may influence the recovery of transformants containinga single copy of the transgene. For example, a lower concentration ofDNA may not necessarily change the efficiency of the transformation butmay instead increase the proportion of single copy insertion events. Inthis regard, ranges of approximately 1 ng to approximately 10 μg (10,000ng), approximately 5 ng to 8 μg or approximately 20 ng, 50 ng, 100 ng,200 ng, 500 ng, 1 μg, 2 μg, 5 μg, or 7 μg of transforming DNA may beused per each 1.0-2.0 mg of starting 1.0 micron gold particles.

Other physical and biological parameters may be varied, such asmanipulation of the DNA/microprojectile precipitate, factors that affectthe flight and velocity of the projectiles, manipulation of the cellsbefore and immediately after bombardment (including osmotic state,tissue hydration and the subculture stage or cell cycle of the recipientcells), the orientation of an immature embryo or other target tissuerelative to the particle trajectory, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.One may particularly wish to adjust physical parameters such as DNAconcentration, gap distance, flight distance, tissue distance, andhelium pressure.

The particles delivered via biolistics can be “dry” or “wet.” In the“dry” method, the mini-chromosome DNA-coated particles such as gold areapplied onto a macrocarrier (such as a metal plate, or a carrier sheetmade of a fragile material such as mylar) and dried. The gas dischargethen accelerates the macrocarrier into a stopping screen, which haltsthe macrocarrier but allows the particles to pass through; the particlesthen continue their trajectory until they impact the tissue beingbombarded. For the “wet” method, the droplet containing themini-chromosome DNA-coated particles is applied to the bottom part of afilter holder, which is attached to a base which is itself attached to arupture disk holder used to hold the rupture disk to the helium egresstube for bombardment. The gas discharge directly displaces the DNA/golddroplet from the filter holder and accelerates the particles and theirDNA cargo into the tissue being bombarded. The wet biolistics method hasbeen described in detail elsewhere but has not previously been appliedin the context of plants (Mialhe et al., Mol Mar Biol Biotechnol.4(4):275-831995). The concentrations of the various components forcoating particles and the physical parameters for delivery can beoptimized using procedures known in the art.

A variety of plant cells/tissues are suitable for transformation,including immature embryos, scutellar tissue, suspension cell cultures,immature inflorescence, shoot meristem, epithelial peels, nodalexplants, callus tissue, hypocotyl tissue, cotyledons, roots, andleaves, meristem cells, and gametic cells such as microspores, pollen,sperm and egg cells. It is contemplated that any cell from which afertile plant may be regenerated is useful as a recipient cell. Callusmay be initiated from tissue sources including, but not limited to,immature embryos, seedling apical meristems, microspore-derived embryos,roots, hypocotyls, cotyledons and the like. Those cells which arecapable of proliferating as callus also are recipient cells for genetictransformation.

Any suitable plant culture medium can be used. Examples of suitablemedia would include but are not limited to MS-based media (Murashige andSkoog, Physiol. Plant, 15:473-497, 1962) or N6-based media(Chu et al.,Scientia Sinica 18:659, 1975) supplemented with additional plant growthregulators including but not limited to auxins such as picloram(4-amino-3,5,6-trichloropicolinic acid), 2,4-D(2,4-dichlorophenoxyacetic acid), naphalene-acetic acid (NAA) anddicamba (3,6-dichloroanisic acid), cytokinins such as BAP(6-benzylaminopurine ) and kinetin, and gibberellins. Other mediaadditives can include but are not limited to amino acids, macroelements,iron, microelements, vitamins and organics, carbohydrates, undefinedmedia components such as casein hydrolysates, an appropriate gellingagent such as a form of agar, a low melting point agarose or Gelrite ifdesired. Those of skill in the art are familiar with the variety oftissue culture media, which when supplemented appropriately, supportplant tissue growth and development and are suitable for planttransformation and regeneration. These tissue culture media can eitherbe purchased as a commercial preparation, or custom prepared andmodified. Examples of such media would include but are not limited toMurashige and Skoog (Mursahige and Skoog, Physiol. Plant, 15:473-497,1962), N6 (Chu et al., Scientia Sinica 18:659, 1975), Linsmaier andSkoog (Linsmaier and Skoog, Physio. Plant., 18:100, 1965), Uchimiya andMurashige (Uchimiya and Murashige, Plant Physiol. 15:473, 1962),Gamborg's B5 media (Gamborg et al., Exp. Cell Res., 50:151, 1968), Dmedium (Duncan et al., Planta, 165:322-332, 1985), Mc-Cown's Woody plantmedia (McCown and Lloyd, HortScience 6:453, 1981), Nitsch and Nitsch(Nitsch and Nitsch, Science 163:85-87, 1969), and Schenk and Hildebrandt(Schenk and Hildebrandt, Can. J. Bot. 50:199-204, 1972) or derivationsof these media supplemented accordingly. Those of skill in the art areaware that media and media supplements such as nutrients and growthregulators for use in transformation and regeneration and other cultureconditions such as light intensity during incubation, pH, and incubationtemperatures can be varied.

Those of skill in the art are aware of the numerous modifications inselective regimes, media, and growth conditions that can be varieddepending on the plant system and the selective agent. Typical selectiveagents include but are not limited to antibiotics such as geneticin(G418), kanamycin, paromomycin or other chemicals such as glyphosate orother herbicides. Consequently, such media and culture conditionsdisclosed in the present invention can be modified or substituted withnutritionally equivalent components, or similar processes for selectionand recovery of transgenic events, and still fall within the scope ofthe present invention.

Mini-Chromosome Delivery Without Selection

Mini-chromosome is delivered to plant cells or tissues, e.g., plantcells in suspension to obtain stably modified callus clones forinheritance assay. Suspension cells are maintained in a growth media,for example Murashige and Skoog (MS) liquid medium containing an auxinsuch as 2,4-dichlorophenoxyacetic acid (2,4-D). Cells are bombardedusing a particle bombardment process, such as the helium-drivenPDS-1000/He system, and propagated in the same liquid medium to penmitthe growth of modified and non-modified cells. Portions of eachbombardment are monitored for formation of fluorescent clusters, whichare isolated by micromanipulation and cultured on solid medium. Clonesmodified with mini-chromosome are expanded and homogenous clones areused in inheritance assays, or assays measuring mini-chromosomestructure or autonomy.

Mini-Chromosome Transformation with Selectable Marker Gene

Isolation of mini-chromosome-modified cells in bombarded calluses orexplants can be facilitated by the use of a selectable marker gene. Thebombarded tissues are transferred to a medium containing an appropriateselective agent for a particular selectable marker gene. Such a transferusually occurs between 0 and about 7 days after bombardment. Thetransfer could also take place any number of days after bombardment. Theamount of selective agent and timing of incorporation of such an agentin selection medium can be optimized by using procedures known in theart. Selection inhibits the growth of non-modified cells, thus providingan advantage to the growth of modified cells, which can be furthermonitored by tracking the presence of a fluorescent marker gene or bythe appearance of modified explants (modified cells on explants may begreen under light in selection medium, while surrounding non-modifiedcells are weakly pigmented). In plants that develop through shootorganogenesis (e.g. Brassica, tomato or tobacco), the modified cells canform shoots directly, or alternatively, can be isolated and expanded forregeneration of multiple shoots transgenic for mini-chromosome. Inplants that develop through embryogenesis (e.g. corn or soybean),additional culturing steps may be necessary to induce the modified cellsto form an embryo and to regenerate in the appropriate media.

Useful selectable marker genes are well known in the art and include,for example, herbicide and antibiotic resistance genes including but notlimited to neomycin phosphotransferase II (conferring resistance tokanamycin, paramomycin and G418), hygromycin phosphotransferase(conferring resistance to hygromycin),5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, conferringresistance to glyphosate), phosphinothricin acetyltransferase(conferring resistance to phosphinothricin/bialophos), MerA (conferringresistance to mercuric ions). Selectable marker genes may be transformedusing standard methods in the art.

The first step in the production of plants containing novel genesinvolves delivery of DNA into a suitable plant tissue (described in theprevious section) and selection of the tissue under conditions thatallow preferential growth of any cells containing the novel genes.Selection is typically achieved with a selectable marker gene present inthe delivered DNA, which may be a gene conferring resistance to anantibiotic, herbicide or other killing agent, or a gene allowingutilization of a carbon source not normally metabolized by plant cells.For selection to be effective, the plant cells or tissue need to begrown on selective medium containing the appropriate concentration ofantibiotic or killing agent, and the cells need to be plated at adefined and constant density. The concentration of selective agent andcell density are generally chosen to cause complete growth inhibition ofwild type plant tissue that does not express the selectable marker gene;but allowing cells containing the introduced DNA to grow and expand intoadchromosomal clones. This critical concentration of selective agenttypically is the lowest concentration at which there is complete growthinhibition of wild type cells, at the cell density used in theexperiments. However, in some cases, sub-killing concentrations of theselective agent may be equally or more effective for the isolation ofplant cells containing mini-chromosome DNA, especially in cases wherethe identification of such cells is assisted by a visible marker gene(e.g., fluorescent protein gene) present on the mini-chromosome.

In some species (e.g., tobacco or tomato), a homogenous clone ofmodified cells can also arise spontaneously when bombarded cells areplaced under the appropriate selection. An exemplary selective agent isthe neomycin phosphotransferase II (nptII) marker gene, which iscommonly used in plant biotechnology and confers resistance to theantibiotics kanamycin, G418 (geneticin) and paramomycin. In otherspecies, or in certain plant tissues or when using particular selectablemarkers, homogeneous clones may not arise spontaneously under selection;in this case the clusters of modified cells can be manipulated tohomogeneity using the visible marker genes present on themini-chromosomes as an indication of which cells contain mini-chromosomeDNA.

Regeneration of Adchromosomal Plants from Explants to Mature, RootedPlants

For plants that develop through shoot organogenesis (e.g. Brassica,tomato and tobacco), regeneration of a whole plant involves culturing ofregenerable explant tissues taken from sterile organogenic callustissue, seedlings or mature plants on a shoot regeneration medium forshoot organogenesis, and rooting of the regenerated shoots in a rootingmedium to obtain intact whole plants with a fully developed root system.These plants are potted in soil and grown to maturity in a greenhouse.

For plant species, such corn and soybean, regeneration of a whole plantoccurs via an embryogenic step that is not necessary for plant specieswhere shoot organogenesis is efficient. In these plants the explanttissue is cultured on an appropriate media for embryogenesis, and theembryo is cultured until shoots form. The regenerated shoots arecultured in a rooting medium to obtain intact whole plants with a fullydeveloped root system. These plants are potted in soil and grown tomaturity in a greenhouse.

Explants are obtained from any tissues of a plant suitable forregeneration. Exemplary tissues include hypocotyls, intemodes, roots,cotyledons, petioles, cotyledonary petioles, leaves and peduncles,prepared from sterile seedlings or mature plants. Brassica tissue can befrom any Brassica species such as Brassica napus, Brassica oleraceae,Brassica nigra, Brassica carinata, Brassica juncea, and Brassicacampestris.

Explants are wounded (for example with a scalpel or razor blade) andcultured on a shoot regeneration medium (SRM) containing Murashige andSkoog (MS) medium as well as a cytokinin, e.g., 6-benzylaminopurine(BA), and an auxin, e.g., α-naphthaleneacetic acid (NAA), and ananti-ethylene agent, e.g., silver nitrate (AgNO₃). For example, 2 mg/Lof BA, 0.05 mg/L of NAA, and 2 mg/L of AgNO₃ can be added to MS mediumfor shoot organogenesis. The most efficient shoot regeneration isobtained from longitudinal sections of internode explants.

Shoots regenerated via organogenesis are rooted in a MS mediumcontaining low concentration of an auxin such as NAA. Plants are pottedand grown in a greenhouse to sexual maturity for seed harvest.

To regenerate a whole plant with a mini-chromosome, explants arepre-incubated for 1 to 7 days (or longer) on the shoot regenerationmedium prior to bombardment with mini-chromosome (see below). Followingbombardment, explants are incubated on the same shoot regenerationmedium for a recovery period up to 7 days (or longer), followed byselection for transformed shoots or clusters on the same medium but witha selective agent appropriate for a particular selectable marker gene(see below).

Method of Co-Delivering Growth Inducing Genes to Facilitate Isolation ofAdchromosomal Plant Cell Clones

Another method used in the generation of cell clones containingmini-chromosomes involves the co-delivery of DNA containing genes thatare capable of activating growth of plant cells, or that promote theformation of a specific organ, embryo or plant structure that is capableof self-sustaining growth. In one embodiment, the recipient cellreceives simultaneously the mini-chromosome, and a separate DNA moleculeencoding one or more growth promoting, organogenesis-promoting,embryogenesis-promoting or regeneration-promoting genes. Following DNAdelivery, expression of the plant growth regulator genes stimulates theplant cells to divide, or to initiate differentiation into a specificorgan, embryo, or other cell types or tissues capable of regeneration.Multiple plant growth regulator genes can be combined on the samemolecule, or co-bombarded on separate molecules. Use of these genes canalso be combined with application of plant growth regulator moleculesinto the medium used to culture the plant cells, or of precursors tosuch molecules that are converted to functional plant growth regulatorsby the plant cell's biosynthetic machinery, or by the genes deliveredinto the plant cell.

The co-bombardment strategy of mini-chromosomes with separate DNAmolecules encoding plant growth regulators transiently supplies theplant growth regulator genes for several generations of plant cellsfollowing DNA delivery. During this time, the mini-chromosome may bestabilized by virtue of its centromere, but the DNA molecules encodingplant growth regulator genes, or organogenesis-promoting,embryogenesis-promoting or regeneration-promoting genes will tend to belost. The transient expression of these genes, prior to their loss, maygive the cells containing mini-chromosome DNA a sufficient growthadvantage, or sufficient tendency to develop into plant organs, embryosor a regenerable cell cluster, to outgrow the non-modified cells intheir vicinity, or to form a readily identifiable structure that is notformed by non-modified cells. Loss of the DNA molecule encoding thesegenes will prevent phenotypes from manifesting themselves that may becaused by these genes if present through the remainder of plantregeneration. In rare cases, the DNA molecules encoding plant growthregulator genes will integrate into the host plant's genome or into themini-chromosome.

As described in Example 3, mini-chromosome DNA has been be co-deliveredinto plant cells together with DNA encoding genes that promote plantcell growth. Under a different embodiment of this invention, the genespromoting plant cell growth may be genes promoting shoot formation orembryogenesis, or giving rise to any identifiable organ, tissue orstructure that can be regenerated into a plant. In this case, it may bepossible to obtain embryos or shoots harboring mini-chromosomes directlyafter DNA delivery, without the need to induce shoot formation withgrowth activators supplied into the medium, or lowering the growthactivator treatment necessary to regenerate plants. The advantages ofthis method are more rapid regeneration, higher transformationefficiency, lower background growth of non-modified tissue, and lowerrates of morphologic abnormalities in the regenerated plants (due toshorter and less intense treatments of the tissue with chemical plantgrowth activators added to the growth medium).

Determination of Mini-Chromosome Structure an Autonomy in AdchromosomalPlants and Tissues

The structure and autonomy of the mini-chromosome in adchromosomalplants and tissues can be determined by methods including but notlimited to: conventional and pulsed-field Southern blot hybridization togenomic DNA from modified tissue subjected or not subjected torestriction endonuclease digestion, dot blot hybridization of genomicDNA from modified tissue hybridized with different mini-chromosomespecific sequences, PCR on DNA from modified tissues with probesspecific to the mini-chromosome, or Fluorescence In Situ Hybridizationto nuclei of modified cells. The table below summarizes these methods.Assay Assay details Potential outcome Interpretation Southern blotRestriction digest of Native sizes and pattern Autonomous or genomicDNA* of bands integrated via CEN compared to purified fragment mini-CAltered sizes or pattern Integrated or rearranged of bands CHEF gelSouthern Restriction digest of Native sizes and pattern Autonomous orblot genomic DNA of bands integrated via CEN compared to purifiedfragment mini-C Altered sizes or pattern Integrated or rearranged ofbands Native genomic DNA Mini-C band migrating Autonomous circles or (nodigest) ahead of genomic DNA linears present in plant Mini-C band co-Integrated migrating with genomic DNA >1 mini-C bands Variouspossibilities observed Exonuclease assay Exonuclease digestion Signalstrength close to Autonomous circles of genomic DNA that w/o exonucleasepresent followed by detection No signal or signal Integrated of circularmini- strength lower that w/o chromosome by PCR, exonuclease dot blot,or restriction digest (optional), electrophoresis and southern blot(useful for circular mini- chromosomes) Mini-chromosome Transformationof Colonies isolated only Autonomous circles rescue plant genomic DNAfrom mini-C plants with present, native mini-C into E. coli followed bymini-Cs, not from structure selection for antibiotic controls; mini-Cresistance genes on structure matches that of mini-C the parental mini-CColonies isolated only Autonomous circles from mini-C plants withpresent, rearranged mini-Cs, not from mini-C structure OR controls;mini-C mini-Cs integrated via structure different from centromerefragment parental mini-C Colonies observed both Various possibilities inmini-C-modified plants and in controls PCR PCR amplification of Allmini-c parts Complete mini-C various parts of the detected by PCRsequences present in mini-chromosome plant Subset of mini-c partsPartial mini-C detected by PCR sequences present in plant FISH Detectionof mini- Mini-C sequences autonomous chromosome sequences detected, freeof genome in mitotic or meiotic Mini-C sequences integrated nuclei byfluorescence detected, associated in situ hybridization with genomeMini-C sequences Both autonomous and detected, both free and integratedmini-C associated with genome sequences present No mini-C sequencesMini-C DNA not visible detected by FISH*Genomic DNA refers to total DNA extracted from plants containing amini-chromosome

Furthermore, mini-chromosome structure can be examined by characterizingmini-chromosomes ‘rescued’ from adchromosomal cells. Circularmini-chromosomes that contain bacterial sequences for their selectionand propagation in bacteria can be rescued from an adchromosomal plantor plant cell and re-introduced into bacteria. If no loss of sequenceshas occurred during replication of the mini-chromosome in plant cells,the mini-chromosome is able to replicate in bacteria and conferantibiotic resistance. Total genomic DNA is isolated from theadchromosomal plant cells by any method for DNA isolation known to thoseskilled in the art, including but not limited to a standardcetyltrimethylammonium bromide (CTAB) based method (Current Protocols inMolecular Biology (1994) John Wiley & Sons, N.Y., 2.3) The purifiedgenomic DNA is introduced into bacteria (e.g., E. coli) using methodsfamiliar to one skilled in the art (for example heat shock orelectroporation). The transformed bacteria are plated on solid mediumcontaining antibiotics to select bacterial clones modified withmini-chromosome DNA. Modified bacterial clones are grown up, the plasmidDNA purified (by alkaline lysis for example), and DNA analyzed byrestriction enzyme digestion and gel electrophoresis or by sequencing.Because plant-methylated DNA containing methylcytosine residues will bedegraded by wild-type strains of E. coli, bacterial strains (e.g. DH10B)deficient in the genes encoding methylation restriction nucleases (e.g.the mcr and mrr gene loci in E. coli) are best suited for this type ofanalysis. Mini-chromosome rescue can be performed on any plant tissue orclone of plant cells modified with a mini-chromosome.

Circular mini-chromosomes that contain bacterial sequences for theirselection and propagation in bacteria can be rescued from anadchromosomal plant or plant cell and re-introduced into bacteria. If noloss of sequences has occurred during replication of the mini-chromosomein plant cells, the mini-chromosome is able to replicate in bacteria andconfer antibiotic resistance. Total genomic DNA is isolated from theadchromosomal plant cells by any method for DNA isolation known to thoseskilled in the art, including but not limited to a standardcetyltrimethylammonium bromide (CTAB) based method (Current Protocols inMolecular Biology (1994) John Wiley & Sons, N.Y., 2.3) The purifiedgenomic DNA is introduced into bacteria (e.g. E. coli) using methodsfamiliar to one skilled in the art (for example heat shock orelectroporation). The transformed bacteria are plated on solid mediumcontaining antibiotics to select bacterial clones modified withmini-chromosome DNA. Modified bacterial clones are grown up, the plasmidDNA purified (by alkaline lysis for example), and DNA analyzed byrestriction enzyme digestion and gel electrophoresis or by sequencing.Because plant-methylated DNA containing methylcytosine residues will bedegraded by wild-type strains of E. coli, bacterial strains (e.g. DH10B)deficient in the genes encoding methylation restriction nucleases (e.g.the mcr and mrr gene loci in E. coli) are best suited for this type ofanalysis. Mini-chromosome rescue can be performed on any plant tissue orclone of plant cells modified with a mini-chromosome.

Mini-Chromosome Autonomy Demonstration by In Situ Hybridization (ISH)

To assess whether the mini-chromosome is autonomous from the nativeplant chromosomes, or has integrated into the plant genome, In SituHybridization is carried out (Fluorescent In Situ Hybridization or FISHis particularly well suited to this purpose). In this assay, mitotic ormeiotic tissue, such as root tips or meiocytes from the anther, possiblytreated with metaphase arrest agents such as colchicines is obtained,and standard FISH methods are used to label both the centromere andsequences specific to the mini-chromosome. For example, for Brassica,the Brassica centromere is labeled using probes from sequence 14F 1,which labels all Brassica chromosomes with one fluorescent tag(Molecular Probes Alexafluor 568, for example), and sequences specificto the mini-chromosome are labeled with another fluorescent tag(Alexafluor 488, for example). All centromere sequences are detectedwith the first tag; only mini-chromosomes are detected with both thefirst and second tag. Chromosomes are stained with a DNA-specific dyeincluding but not limited to DAPT, Hoechst 33258, OliGreen, Giemsa YOYO,and TOTO. An autonomous mini-chromosome is visualized as a body thatshows hybridization signal with both centromere probes andmini-chromosome specific probes and is separate from the nativechromosomes. Similar procedures can be carried out for centromeresderived from other plant species.

Determination of Gene Expression Levels

The expression level of any gene present on the mini-chromosome can bedetermined by methods including but not limited to one of the following.The mRNA level of the gene can be determined by Northern Blothybridization, Reverse Transcriptase-Polymerase Chain Reaction, bindinglevels of a specific RNA-binding protein, in situ hybridization, or dotblot hybridization.

The protein level of the gene product can be determined by Western blothybridization, Enzyme-Linked Immunosorbant Assay (ELISA), fluorescentquantitation of a fluorescent gene product, enzymatic quantitation of anenzymatic gene product, immunohistochemical quantitation, orspectroscopic quantitation of a gene product that absorbs a specificwavelength of light.

Use of Exonuclease to Isolate Circular Mini-Chromosome DNA from GenomicDNA:

Exonucleases may be used to obtain pure mini-chromosome DNA, suitablefor isolation of mini-chromosomes from E. coli or from plant cells. Themethod assumes a circular structure of the mini-chromosome. A DNApreparation containing mini-chromosome DNA and genomic DNA from thesource organism is treated with exonuclease, for example lambdaexonuclease combined with E. coli exonuclease I, or the ATP-dependentexonuclease (Qiagen Inc). Because the exonuclease is only active on DNAends, it will specifically degrade the linear genomic DNA fragments, butwill not affect the circular mini-chromosome DNA. The result ismini-chromosome DNA in pure form. The resultant mini-chromosome DNA canbe detected by a number of methods for DNA detection known to thoseskilled in the art, including but not limited to PCR, dot blot followedby hybridization analysis, and southern blot followed by hybridizationanalysis. Exonuclease treatment followed by detection of resultantcircular mini-chromosome may be used as a method to determinemini-chromosome autonomy.

Structural Analysis of Mini-Chromosomes by BAC-End Sequencing:

BAC-end sequencing procedures, known to those skilled in the art, can beapplied to characterize mini-chromosome clones for a variety ofpurposes, such as structural characterization, determination of sequencecontent, and determination of the precise sequence at a unique site onthe chromosome (for example the specific sequence signature found at thejunction between a centromere fragment and the vector sequences). Inparticular, this method is useful to prove the relationship between aparental mini-chromosome and the mini-chromosomes descended from it andisolated from plant cells by mini-chromosome rescue, described above.

Methods for Scoring Meiotic Mini-Chromosome Inheritance

A variety of methods can be used to assess the efficiency of meioticmini-chromosome transmission. In one embodiment of the method, geneexpression of genes encoded by the mini-chromosome (marker genes ornon-marker genes) can be scored by any method for detection of geneexpression know to those skilled in the art, including but not limitedto visible methods (e.g. fluorescence of fluorescent protein markers,scoring of visible phenotypes of the plant), scoring resistance of theplant or plant tissues to antibiotics, herbicides or other selectiveagents, by measuring enzyme activity of proteins encoded by themini-chromosome, or measuring non-visible plant phenotypes, or directlymeasuring the RNA and protein products of gene expression usingmicroarray, northern blots, in situ hybridization, dot blothybridization, RT-PCR, western blots, immunoprecipitation, Enzyme-LinkedImmunosorbant Assay (ELISA), immunofluorescence and radio-immunoassays(RIA). Gene expression can be scored in the post-meiotic stages ofmicrospore, pollen, pollen tube or female gametophyte, or thepost-zygotic stages such as embryo, seed, or progeny seedlings andplants. In another embodiment of the method, the mini-chromosome can dedirectly detected or visualized in post-meiotic, zygotic, embryonal orother cells in by a number of methods for DNA detection known to thoseskilled in the art, including but not limited to fluorescence in situhybridization, in situ PCR, PCR, southern blot, or by mini-chromosomerescue described above.

FISH Analysis of Mini-Chromosome Copy Number in Meiocytes, Roots orOther Tissues of Adchromosomal Plants

The copy number of the mini-chromosome can be assessed in any cell orplant tissue by In Situ Hybridization (Fluorescent In Situ Hybridizationor FISH is particularly well suited to this purpose). In an exemplaryassay, standard FISH methods are used to label the centromere (e.g., forBrassica, using probes from sequence 14F1 which labels all Brassicachromosomes with one fluorescent tag (Molecular Probes Alexafluor 568,for example)), and to label sequences specific to the mini-chromosomewith another fluorescent tag (Alexafluor 488, for example). Allcentromere sequences are detected with the first tag; onlymini-chromosomes are detected with both the first and second tag. Nucleiare stained with a DNA-specific dye including but not limited to DAPI,Hoechst 33258, OliGreen, Giemsa YOYO, and TOTO. Mini-chromosome copynumber is determined by counting the number of fluorescent foci thatlabel with both tags.

Induction of Callus and Roots from Adchromosomal Plants Tissues forInheritance Assays

Mini-chromosome inheritance is assessed using callus and roots inducedfrom transformed plants. To induce roots and callus, tissues such asleaf pieces are prepared from adchromosomal plants and cultured on aMurashige and Skoog (MS) medium containing a cytokinin, e.g.,6-benzylaminopurine (BA), and an auxin, e.g., α-naphthaleneacetic acid(NAA). Any tissue of an adchromosomal plant can be used for callus androot induction, and the medium recipe for tissue culture can beoptimized using procedures known in the art.

Clonal Propagation of Adchromosomal Plants

To produce multiple clones of plants from a mini-chromosome-transformedplant, any tissue of the plant can be tissue-cultured for shootorganogenesis using regeneration procedures described under the sectionregeneration of plants from explants to mature, rooted plants (seeabove). Alternatively, multiple auxiliary buds can induced from amini-chromosome-modified plant by excising the shoot tip, which can berooted for a whole plant; each auxiliary bud can be rooted for a wholeplant.

Scoring of Antibiotic- or Herbicide Resistance in Seedlings and Plants(Progeny of Self- and Out-Crossed Transformants

Progeny seeds harvested from mini-chromosome-modified plants can bescored for antibiotic- or herbicide resistance by seed germination understerile conditions on a growth media (for example Murashige and Skoog(MS) medium) containing an appropriate selective agent for a particularselectable marker gene. Only seeds containing the mini-chromosome cangerminate on the medium and further grow and develop into whole plants.Alternatively, seeds can be germinated in soil, and the germinatingseedlings can then be sprayed with a selective agent appropriate for aselectable marker gene. Seedlings that do not contain mini-chromosome donot survive; only seedlings containing mini-chromosome can survive anddevelop into mature plants.

Genetic Methods for Analyzing Mini-Chromosome Performance:

In addition to direct transformation of a plant with a mini-chromosome,plants containing a mini-chromosome can be prepared by crossing a firstplant containing the functional, stable, autonomous mini-chromosome witha second plant lacking the construct.

Fertile plants modified with mini-chromosomes can be crossed to otherplant lines or plant varieties to study mini-chromosome performance andinheritance. In the first embodiment of this method, pollen from anadchromosomal plant can be used to fertilize the stigma of anon-adchromosomal plant. Mini-chromosome presence is scored in theprogeny of this cross using the methods outlines in the precedingsection. In the second embodiment, the reciprocal cross is performed byusing pollen from a non-adchromosomal plant to fertilize the flowers ofa adchromosomal plant. The rate of mini-chromosome inheritance in bothcrosses can be used to establish the frequencies of meiotic inheritancein male and female meiosis. IP the third embodiment of this method, theprogeny of one of the crosses just described are back-crossed to thenon-adchromosomal parental line, and the progeny of this second crossare scored for the presence of genetic markers in the plant's naturalchromosomes as well as the mini-chromosome. Scoring of a sufficientmarker set against a sufficiently large set of progeny allows thedetermination of linkage or co-segregation of the mini-chromosome tospecific chromosomes or chromosomal loci in the plant's genome. Geneticcrosses performed for testing genetic linkage can be done with a varietyof combinations of parental lines; such variations of the methodsdescribed are known to those skilled in the art.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention andwithout diminishing its intended advantages. It is therefore intendedthat such changes and modifications be covered by the appended claims.

EXAMPLE 1 Brassica Centromere Construction

BAC Library Construction

A Bacterial Artificial Chromosome (BAC) library was constructed fromBrassica genomic DNA isolated from B. oleraceae variety “Wisconsinfastplants” and digested with the restriction enzyme MboI. This enzymewas chosen because it is methylation insensitive and therefore can beused to enrich BAC libraries for centromere DNA sequences.

Probe Identification and Selection

Three groups of Brassica repetitive genomic DNA including specificcentromere-localized sequences, were initially compiled as candidateprobes for hybridization with the BAC libraries (Table 3). These probesrepresented various classes of Brassica repetitive sequences includingsatellite repeats (heterochromatic/centromere-specific), rDNA, andhypermethylated DNA fractions.

Four probes were picked to interrogate the BAC libraries. These probesrepresented different groups of commonly found repetitive sequences inthe Brassica genome. The probes selected (Table 3) were CANREP (theBrassica centromere satellite), rRNA (18S), HpaII (bulk methylated DNApurified from genomic DNA by failure to digest with themethylation-sensitive enzyme HpaII) and Sau3A (bulk methylated DNApurified from genomic DNA by failure to digest with themethylation-sensitive enzyme Sau3A1). The probes were prepared fromcloned fragments or from bulk methylated DNA prepared from Brassicagenomic DNA. Sequences from the clones used to prepare each probe areshown in Table 3. Probes were prepared and labeled with standardmolecular biology methods. TABLE 3 Brassica repetitive genomic sequenceand BAC library probes Group GenBank Group # Name Probe Name DescriptionClone used for hyb accession* Reference or comment 1 rDNA B6A10 18S rRNA5012-5-6-A10 AF207007.1 (SEQ ID NO: 27) 2 Cen BF1 CANREP 5012-5-14-F01X68786.1 CANREP is one of a large family of repeat (SEQ ID NO: 28)sequences hit by this sequence: gi|860800|emb|X68786.1|BJCANRD B. junceaXle7-2EB gene gi|860798|emb|X68784.1|BJCANRB B. juncea Xle4-7B genegi|17706|emb|X12736.1|BCREPC Brassica campestris DNA for satellite 3Bulk Bhpaii Purified N/A N/A repetitive methylated DNA DNA fraction BsauPurified N/A N/A methylated DNA fraction*Accession number of BLAST hit; actual sequence has not been depositedin GenbankLibrary Interrogation and Data Analysis

The BAC clones from the libraries were spotted onto filters and thefilters were hybridized with each of the probes to identify specific BACclones that contain DNA from the group of sequences represented by theprobe(s).

A total of 18,432 BAC clones from the library were interrogated witheach of the probes described above using the following hybridizationconditions: 0.5×SSC 0.25% SDS at 65 degrees for 15 minutes, followed bya wash at 65 degrees for a half hour. The hybridization intensities ofthe BAC clones with each probe were scanned to quantitate hybridizationintensity for each clone. The outputs (scores of 1 to 10 based on thehybridization intensities, with 10 being the strongest hybridizationintensity) were imported into a relational database, for furtheranalysis and classification. The database contained a total of fourtables. Each table contained at total of 18,432 entries: thehybridization scores of each BAC clone from the library to one of theprobes used to interrogate the library. Data analysis was done usingstandard SQL (Structured Query Language) routines to find BACs thatcontain different groups of repetitive sequences.

Classification and Selection of BAC Clones for Mini-ChromosomeConstruction

BAC clones containing centromeric/heterochromatic DNA were identified bytheir hybridization scores to different probes. The goal was to selectBAC clones that contained a diverse set of various repetitive sequences.Nine classes of centromeric BAC clones were eventually chosen to coverthe broadest possible range of centromeric/heterochromatic sequences formini-chromosome construction. Detailed descriptions of each class andprobe hybridization values for each class are shown in Table 4. TABLE 4Classification of Brassica BAC clones containing centromeric DNA ClassProbe Hybridization Range* # clones Class Properties CANREP HpaII Sau3ArDNA identified A Hi CANREP >=7 >=7 >=7 N/A 33 Hi Sau + Hpa B HiCANREP >=7 N/A >=7 <=4 7 and Sau, Low rDNA C Hi Sau and N/A >=8 >=8 N/A43 Hpa D Hi CANREP >=8 >=7 N/A N/A 123 and Hpa E Hi CANREP >=8 N/A >=7N/A 59 and Sau F Hi CANREP >=7 <=4 <=4 N/A 15 only G Hi Sau only <=4<=4 >=7 N/A 8 H Hi Hpa only <=4 >=7 <=4 N/A 58 I Hi >=7 4 to 6 4 to 6N/A 210 CANREP, middle meth Total** 556*Values represent hybridization intensities of an individual BAC to eachprobe on a scale of 1 to 10. Values were normalized.N/A = not applicable

A number of representative clones from each class were chosen to yield atotal of 190 BAC clones for further analysis by restriction digestfingerprinting. The BAC clones were fingerprinted based on restrictionsites found in the centromere specific sequence(s). Fingerprinting wasused to evaluate the sequence composition of the large numbers of BACclones and to compare their similarity to each other by comparing therestriction enzyme digest fragment patterns. A sequence with a tandemrepeated sequence will show a single intense band of unit repeat sizewhen digested with a restriction enzyme that cuts within the unitrepeat. Second, BAC clones with similar sequences will show similarpatterns of restriction fragments in a digest.

BAC DNA was extracted from bacteria using methods familiar to thoseskilled in the art. For Brassica, the restriction enzyme HindIII wasused to digest the BAC clones. Colonies containing the BAC clones weregrown overnight at 37° C. with shaking at 250-300 rpm. DNA from thecolonies was isolated using Qiagen solution PI, Qiagen solution P2,Qiagen solution P3, followed by phenol/chloroform extraction.Subsequently, 10 μl of each DNA sample was inserted in into a well on a96-well plate. The DNA samples were mixed with 10 μl of the followingmixture: 200 μl 10× buffer (New England Biolabs), 50 ul 100× BSA (NewEngland Biolabs), 30 μl enzyme (varies depending on Class of BAC clone)and 750 μl water. The samples were covered and incubated at 37° C. 1-4hours. After the incubation, loading dye was added to each sample andthe DNA was analyzed on a 1% agarose gel in 1× TBE, 23volts for 14-18hours.

For Brassica, the restriction enzyme HindIII was used to digest the BACclones. After fingerprinting, 100 BACs were selected based on thefingerprint analysis in order to represent the hybridization classes,with an emphasis on the different classes containing the centromeretandem repeat. Within the hybridization classes, fingerprints showingthe ‘simple ladder’ of ‘complex ladder’ patterns of bands at integermultiples of the unit centromere tandem repeat were favored.Additionally, within the hybridization classes, BAC clones thatrepresent the diversity of fingerprints were preferred. Also, cloneswith matching fingerprints were not chosen. In some cases, after a roundof functional testing, additional BACs were selected for theirsimilarity of hybridization class and fingerprint to a BAC that showedgood centromere function.

Twenty five BAC clones (from the original 190) were selected formini-chromosome construction based on the fingerprint class. These BACsare listed in Table 5. Fingerprints were classified into 3 classes: 1.high complexity (multiple large bands with no indication of laddering),2. low ladder (predominant bands at multiples of the unit repeat sizefor the centromere satellite, and 3. complex ladder (features of bothprevious types). Subsequent to testing, 4 additional BACs (BB221, BB222,BB229 and BB280) were chosen from the library based on their similarityto BB5 in both hybridization pattern and fingerprint. The preferred BACShave an *. Table 6 lists the fingerprint classes for 11 selectedBrassica BACs. TABLE 5 Restriction endonuclease fingerprinting of 25Brassica BACs BAC BAC Hind III MiniC Number Class Class PropertiesFingerprint Class tested BB2 A Hi CANREP, Meth 3. Complex ladder BB2R1-1BB5* A Hi CANREP, Meth 3. Complex ladder BB5R4-1 BB5R4-3 BB7 B HiCANREP, Meth, 1. Complex BB7R2-1 low rDNA BB11 D Hi CANREP, Meth 2.Simple ladder BB11R1-2 (Hpa) BB15 C Hi Meth 3. Complex ladder BB15R4-1BB16* D Hi CANREP, Meth 1. Complex BB16R1-2 (Hpa) BB16R1-3 BB18* D HiCANREP, Meth 1. Complex BB18R1-2 (Hpa) BB18R2-3 BB38* F Hi CANREP only3. Complex ladder BB38R1-3 BB39 C Hi Meth n/d* BB39R1-3 BB40 C Hi Methn/d* BB40R1-2 BB40R1-3 BB40R2-1 BB40R3-1 BB47* D Hi CANREP, Meth 2.Simple ladder BB47R1-2 (Hpa) BB52 E Hi CANREP, Meth 1. Complex BB52R1-1(Sau) BB60* D Hi CANREP, Meth 3. Complex ladder BB60R1-1 (Hpa) BB63* DHi CANREP, Meth 2. Simple ladder BB63R1-1 (Hpa) BB64 I Hi CANREP, 1.Complex BB64R1-1 Moderate Meth BB70* I Hi CANREP, 2. Simple ladderBB70R1-3 Moderate Meth BB71* E Hi CANREP, Meth 3. Complex ladderBB71R1-1 (Sau) BB76* I Hi CANREP, 1. Complex BB76R1-3 Moderate MethBB102 D Hi CANREP, Meth n/d* BB102R1-1 (Hpa) BB104* I Hi CANREP, n/d*BB104R1-2 Moderate Meth BB105 I Hi CANREP, 2. Simple ladder BB105R1-2Moderate Meth BB106 D Hi CANREP, Meth 2. Simple ladder BB106R1-2 (Hpa)BB119 I Hi CANREP, 3. Complex ladder BB119R1-1 Moderate Meth BB129 D HiCANREP, Meth n/d* BB129R1-1 (Hpa) BB140 A Hi CANREP, Meth 2. Simpleladder BB140R1-3 BB221 A Hi CANREP, Meth 3. Complex ladder BB221R2-1BB222 A Hi CANREP, Meth 3. Complex ladder BB222R2-7 BB229 A Hi CANREP,Meth 3. Complex ladder BB229R2-6 BB280 A Hi CANREP, Meth 3. Complexladder BB280R2-3n/d*: Gel too faint to score

TABLE 6 Restriction endonuclease fingerprint classification for 11selected Brassica BACs BAC Hind III fingerprint Number Class ClassProperties class BB5 A Hi CANREP, Meth 3. Complex ladder BB16 D HiCANREP, Meth (Hpa) 1. Complex BB18 D Hi CANREP, Meth (Hpa) 1. ComplexBB38 F Hi CANREP only 3. Complex ladder BB47 D Hi CANREP, Meth (Hpa) 2.Simple ladder BB60 D Hi CANREP, Meth (Hpa) 3. Complex ladder BB63 D HiCANREP, Meth (Hpa) 2. Simple ladder BB70 I Hi CANREP, Moderate 2. Simpleladder Meth BB71 E Hi CANREP, Meth (Sau) 3. Complex ladder BB76 I HiCANREP, Moderate 1. Complex Meth BB104 I Hi CANREP, Moderate n/d* Methn/d*: Gel too faint to score

B. oleraceae (broccoli) BAC BB5 was deposited with the American TypeCulture Collection (ATCC) P.O. Box 1549 Manassas, Va. 20108, USA on Feb.23, 2005 and assigned Accession No. ______.

To determine the molecular weight of centromere fragments in the BAClibraries, a frozen sample of bacteria harboring a BAC clone was grownin selective liquid media and the BAC DNA harvested using a standardalkaline lysis method. The recovered BAC DNA was restriction digestedand resolved on an agarose gel. Centromere fragment size was determinedby comparing to a molecular weight standard.

Cre/lox recombined donor DNA and BAC centromere DNA was delivered intobacteria and plated on selective solid media. To determine the molecularweight of centromere fragments in retrofitted mini-chromosomes, threebacterial colonies harboring a mini-chromosome were independently grownin selective liquid media and the BAC DNA harvested using a standardalkaline lysis method. The recovered BAC DNA was restriction digestedand resolved on an agarose gel. Centromere fragment size was determinedby comparing to a molecular weight standard. If variation in centromeresize was noted, the mini-chromosome with the largest centromere insertwas used for further experimentation.

EXAMPLE 2 Assembly and Components of Brassica Mini-Chromosomes

Two methods have been developed to construct plant mini-chromosomes. Thefirst method relies on cre/lox recombination in which a bacterialmini-chromosome (BAC) vector containing plant centromeric DNA and a loxPrecombination site is recombined, by the action of cre recombinase, witha donor vector carrying plant gene expression cassettes to generate aplant mini-chromosome. The second method uses restriction enzymedigestion and ligation to produce two DNA fragments with compatiblecohesive ends: 1) a vector fragment containing plant gene expressioncassettes and ii) a centromere fragment. The two fragments are ligatedinto a circular structure to form a plant mini-chromosomes.

The components of the Brassica mini-chromosomes include fluorescentreporter genes, a selectable maker gene, a Brassica centromere sequenceidentified in a Brassica BAC library, a telomere sequence, a cloningvector and a donor vector. These components are described in detailbelow.

Mini-Chromosome Construction by Cre-Lox Recombination

Cre recombinase-mediated exchange was used to construct mini-chromosomesby combining the plant centromere fragments cloned in pBeloBAC11 with adonor plasmid (e.g. pCHR151, Table 10). The recipient BAC vectorcarrying the plant centromere fragment contained a loxP recombinationsite; the donor plasmid contained two such sites, flanking the sequencesto be inserted into the recipient BAC. Mini-chromosomes were constructedusing a two-step method. First, the donor plasmid was linearized toallow free contact between the two loxP site; in this step the backboneof the donor plasmid is eliminated. In the second step, the donormolecules were combined with centromere BACs and were treated with Crerecombinase, generating circular mini-chromosomes with all thecomponents of the donor and recipient DNA. Mini-chromosomes weredelivered into E. coli and selected on medium containing kanamycin andchloramphenicol. Only vectors that successfully cre recombined andcontained both selectable markers survived in the medium.Mini-chromosomes were extracted from bacteria and restriction digestedto verify DNA composition and calculate centromere insert size (Table7). TABLE 7 Cre/Lox Recombined Mini-chromosomes forBrassica BrassicaCentromere Centromere insert Mini-Chromosome Fragment (kbp) Donor VectorBB5R4-1 5 64 pCHR151 BB5R10-1 5 48 pCHR171A BB5R14-6 5 52 pCHR487BB5R15-4 5 52 pCHR488 BB5R16-6 5 50 pCHR489 BB71R1-1 71 30 pCHR151BB221R2-1 221 70 pCHR487 BB222R2-7 222 60 pCHR487 BB229R2-6 229 60pCHR487 BB280R2-3 280 97 pCHR487Mini-Chromosome Construction by Restriction-Ligation

Mini-chromosomes were also constructed using standard cloningprocedures. For example, a BAC containing a centromere fragment wasdigested with a restriction endonuclease that created sticky ends, asfor example, but not limited to NotI, which was commonly used for thispurpose. The digested DNA was then electrophoresed to purify thecentromere fragment into a single band. The electrophoresis was carriedout with either conventional agarose gel electrophoresis with a linearelectric field, or CHEF gel electrophoresis using an electric field thatswitches its orientation in the course of the run. When theelectrophoresis was complete, the centromere fragment was visualized byethidium bromide staining and illumination under ultraviolet light. Theband corresponding to centromere DNA was excised, and the DNA waspurified from the gel using conventional method for gel-purifying DNAfragments from agarose gels. The purified fragment was then ligated witha vector fragment that contains a low-copy E. coli backbone (e.g. the F′plasmid replicon) and one or more plant-expressed genes. The vectorfragment was digested with a restriction endonuclease leaving compatiblesticky ends to those present on the centromere fragment. Alternatively,both fragments may be blunt.

To achieve a high rate of insertion of the centromere fragment into thevector, the phosphate groups were removed from the ends of the vectormolecule by treating this DNA molecule with phosphatase; this stepprevented ligation of the vector molecule to itself or to other vectormolecules. After ligating vector DNA and centromere fragment, themini-chromosomes were delivered into E. coli and selected on mediumcontaining antibiotics corresponding to the antibiotic-resistance genespresent on the vector molecule (e.g. kanamycin and chloramphenicol).Mini-chromosomes are extracted from bacteria and restriction digested toverify DNA composition and calculate centromere insert size (Table 8).TABLE 8 Restriction-Ligation Mini-chromosomes Brassica CentromereCentromere insert Mini-Chromosome Fragment (kbp) Donor Vector pCHR5435R4-1 64 pCHR510 pCHR591 5R4-1 64 pCHR579 pCHR593 5R4-1 64 pCHR581pCHR816 5R4-1 64 pCHR806 pCHR817 5R4-1 64 pCHR807 pCHR818 5R4-1 64pCHR808 pCHR819 5R4-1 64 pCHR809 pCHR820 5R4-1 64 pCHR810 pCHR821 5R4-164 pCHR811 pCHR823 5R4-1 64 pCHR813 pCHR824 5R4-1 64 pCHR814 pCHR8255R4-1 64 pCHR815 pCHR955 5R4-1 64 pCHR945 pCHR958 5R4-1 64 pCHR948pCHR964 15R4-1 121 pCHR807 pCHR965 15R4-1 121 pCHR815 pCHR967 16R1-2 156pCHR815 pCHR970 52R1-1 99 pCHR807 pCHR972 60R1-1 49 pCHR807 pCHR97360R1-1 49 pCHR815Cloning Vector

The vector, pBeloBAC11, is an E. coli plasmid cloning vector based onthe F′ plasmid replicon of E. coli. The vector contained achloramphenicol resistance gene for selection of the plasmid inbacteria, repE, sopA/B/and C for maintenance of the plasmid in bacteria,and a LoxP recombination site for specific cleavage by Cre recombinase.A description of all the genes contained within the vector and thelocation of the gene within the vector are set out in Table 9. TABLE 9pBeloBAC11 components Size (base Genetic Element pair) Location (bp)Details Bacterial 660  766-1425 Bacterial selectable Chloramphenicol(complementary) marker resistance ori2 67 2370-2436 F′ plasmid origin ofreplication from E. coli repE 755 2765-3520 mediation of replicationcomplex at Ori2 (Mori, H et. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15)SopA 1166 4108-5274 partition of plasmid to bacterial daughter cells(Mori, H et. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15) SopB 9715274-6245 partition of plasmid to bacterial daughter cells (Mori, H et.al, J Mol Biol. 1986 Nov 5; 192(1): 1-15) SopC 474 6318-6791 partitionof plasmid to bacterial daughter cells (Mori, H et. al, J Mol Biol. 1986Nov 5; 192(1): 1-15) cos 400 7050-7449 Lambda DNA recognition sequencefor phage packaging LoxP 34 7467-7500 Recombination site for Cremediated recombination (Arenski et. al 1983, Abremski et. al 1984)Source of Coding Regions Used in Plant-Expressed Genes

The fluorescent reporter genes DsRed, and AmCyan were isolated fromAthozoa species; and ZsYellow and ZsGreen were isolated from Zoanthus sp(Matz et. al. Nature Biotechnol. 1999 October; 17:969). These reportergenes express proteins that are homologous to Green Fluorescent Protein(GFP), which is a commonly used reporter gene in various biologicalsystems, including plants. All fluorescent reporter genes were obtainedfrom Clontech Corporation (Palo Alto, Calif.).

The selectable marker gene MerA is a mercuric ion reductase whichconverts toxic Hg²⁺ to less toxic metallic mercury. This gene wasoriginally isolated from E. coli and then modified to accommodateimproved expression in plants (Rugh et. al. PNAS 1996 93:318).

The selectable marker gene NPTII (neomycin phosphotransferase II) hasbeen commonly used in plants as a selectable agent (Bevan et.al. Nature1983 304:184). The original source of this gene is E. coli.

Donor Vectors Used to Construct Mini-Chromosomes via Cre/LoxRecombinations

pCHR151

The plasmid pCHR151 was developed using the commercially available highcopy number E. coli cloning vector pUC19 (Yanisch-Perron et al., (1985)Gene 33, 103-119). The plasmid backbone was modified with the bacterialkanamycin selectable marker for maintenance of the plasmid in bacterialhosts, a pair of complementary loxP sites and a polylinker thatfacilitated the modular assembly of several plant-expressed genes forexpression in plant mini-chromosomes. Using standard cloning methods,plant-expressed gene cassettes were introduced into the modified pUC19vector to construct pCHIR151. This vector includes DsRed with a nuclearlocalization signal (Clontech Corporation, Palo Alto Calif.), which wasregulated by the Arabidopsis UBQ10 promoter (At4g05320) and theArabidopsis pyruvate kinase terminator (At5g52920). The vector alsoincluded the E. coli MerA gene regulated by the Arabidopsis thalianaACT2 promoter and terminator. The vector also contains a high-copy E.coli replication origin and an ampicillin bacterial selectable marker.Mini-chromosome genetic elements within the pCHR151 vector are set outin Table 10.

Prior to using pCHR151 to construct plant mini-chromosomes, pCHR151 wasdigested with restriction endonucleases to linearize the pCHR151 plasmidand remove the high copy origin of replication and the bacterialampicillin selectable marker, leaving loxP recombination sites on eachend of the linear fragment. The resulting linearized vector was crerecombined in vitro to generate circular donor pCHR151 plasmids lackinga bacterial origin of replication and the ampicillin selectable marker.The donor pCHR151 construct was used to construct plantmini-chromosomes. TABLE 10 Donor Components of pCHR151 Size GeneticElement (bp) Location (bp) Details Act2 promoter + 1482 7473-8954 TheArabidopsis thaliana intron (complementary) promoter Actin 2 plusnatural intron. MerA 1695 5776-7470 Plant selectable marker(complementary) providing resistance to mercuric ions (Rugh et.al. PNAS1996 93: 3182). Act2 800 4823-5622 Arabidopsis thaliana Actin terminator(complementary) 2 terminator. Bacterial 817 3825-4641 Bacterialkanamycin Kanamycin (complementary) selectable marker Pyruvate 3323349-3680 Arabidopsis thaliana kinase Pyruvate kinase terminatorterminator (At5g52920) DsRed2 + 780 2435-3214 Nuclear localized red NLSfluorescent protein from Discosoma sp. (Matz, M et. al Nat Biotechnol1999 Dec; 17(12): 1227). UBQ10 2038  361-2398 Arabidopsis thalianaPromoter polyubiquitin promoter (At4g05320) LoxP 34 346-379Recombination site for Cre and mediated recombination 9005-9038 (Arenskiet. al 1983, Abremski et. al 1984)

pCHR171A

The vector pCHR171A vector was used to generate linear mini-chromosomesby introducing plant telomere sequences. The donor region of pCHR171A isidentical to pCHR151 (described above) with the exception of two planttelomeric regions located on both sides of the bacterial kanamycin gene.pCHR171A was constructed using standard cloning methods. Similar toconstruction of pCHR151, the low copy bacterial backbone of pBeloBAC11was used in place of the pUC19 high copy backbone to stabilize theaddition of the highly repetitive plant telomeric sequences. Thebacterial tetracycline gene replaced the pBeloBAC11 chloramphenicol genefor bacterial selection.

Naturally occurring plant telomeres are composed of a seven nucleotiderepeat (TAAACCC). Plant telomeres were polymerized using standard PCRmethods to generate approximately 800 base pair telomere arrays. Thetelomere sequences were ligated using standard methods on both sides ofthe bacterial kanamycin gene. Two unique I-PpoI homing endonucleaserestriction sites were introduced between each telomere and thekanamycin gene for linearization of the final mini-chromosome construct.Mini-chromosome genetic elements within the pCHR171 vector are set outin Table II below. TABLE 11 Donor Components of pCHR171A Size GeneticElement (base pair) Location (bp) Details Act2 promoter + intron 1482 97-1578 The Arabidopsis thaliana promoter Actin 2 plus natural intron.MerA 1695 1581-3275 Plant selectable marker providing resistance tomercuric ions (Rugh et.al. PNAS 1996 93: 3182). Act2 terminator 8003429-4228 Arabidopsis thaliana Actin 2 terminator. Plant telomere 7594277-5035 Plant telomere PCR based on plant consensus telomere sequenceBacterial Kanamycin 817 5211-6027 Bacterial kanamycin selectable markerPlant telomere 760 6161-6920 Plant telomere PCR based on plant consensustelomere sequence Pyruvate kinase 332 6968-7299 Arabidopsis thalianaPyruvate terminator kinase terminator (At5g52920) DsRed2 + NLS 7807434-8213 Nuclear localized red fluorescent protein from Discosoma sp.(Matz, M et. al Nat Biotechnol 1999 Dec; 17(12): 1227). UBQ10 Promoter2038  8250-10287 Arabidopsis thaliana polyubiquitin promoter (At4g05320)LoxP 34 47-80 and 10303-10336 Recombination site for Cre mediatedrecombination (Arenski et. al 1983, Abremski et. al 1984)

To generate plant mini-chromosomes with pCHR171A, the vector wasdigested and cre treated using the same methods as described for pCHR151to generate donor pCHR171A. Restriction digests of pCHR171A removed thelow copy origin of replication and the bacterial tetracycline selectablemarker, leaving loxP recombination sites on each end of the linearfragment. The resulting linearized vector was cre recombined in vitro togenerate circular donor pCHR171A plasmids lacking a bacterial origin ofreplication and the tetracycline selectable marker.

Cre recombinase-mediated exchange was used to construct mini-chromosomesby combining the plant centromere fragments of pBeloBAC11 with the donorvector pCHR171A. The recipient BAC vector carrying the plant centromerefragment contained a loxP recombination site, facilitating theintroduction of donor DNA via the action of cre recombinase. Usingpurified cre recombinase in vitro, BAC centromere recipients werecombined with donor pCHR171A DNA, generating circular mini-chromosomeswith all the components of the donor and recipient DNA. Mini-chromosomeswere delivered into E. coli and selected on medium containing kanamycinand chloramphenicol. Only vectors that successfully cre recombinedcontained both selectable markers and were easily selected fromnon-recombined events. Mini-chromosomes were extracted from bacteria andrestriction digested to verify DNA composition and calculate centromereinsert size.

To generate linear mini-chromosomes constructed with donor pCHR171A, E.coli harboring the mini-chromosomes were grown in selective bacterialgrowth medium and purified using standard alkaline lysis procedures.Purified mini-chromosomes were restriction digested in vitro with homingendonuclease enzyme I-PpoI following standard restriction digestprocedures. Linearization of the mini-chromosome results in the removalof the bacterial kanamycin gene cassette leaving plant telomeresflanking both ends of the linear mini-chromosome. Linearmini-chromosomes were ethanol precipitated and used for planttransformation.

Other Donor Vectors Used Via Cre/Lox Recombination

The pCHR487 mini-chromosome donor vector was also used to generateBrassica mini-chromosomes. In this vector, the Act2 promoter-MerA genecassette of pCHR151 was replaced with the yeast TEF2 promoter fromSaccharomyces cerevisiae and the plant kanamycin selectable marker NptIIfrom E. coli. To enhance the stability of the NptII transcript, theArabidopsis thaliana UBQ10 intron was inserted 5′ of the yeast TEF2promoter and 3′ of the NptlI gene. The UBQ10 intron is a naturallyoccurring component of the transcribed sequences from the Arabidopsisthaliana UBQ10 gene and was present in the UB10 promoter in pCHR151.Standard restriction digest and cloning methods were used to generatepCHR487. Construction of plant mini-chromosomes using pCHR487 wasperformed as described for pCHR151. As with pCHR151, the circular donorpCHR487 lacked a bacterial origin of replication and the bacterialampicillin selectable marker. Mini-chromosome genetic elements withinthe pCHR487 vector are set out in Table 12. TABLE 12 Donor Components ofpCHR487 Size Genetic Element (base pair) Location (bp) Details UBQ10promoter 2038 361-2398 Arabidopsis thaliana polyubiquitin promoter(At4g05320) DsRed2 + NLS 780 2435-3214 Nuclear localized red fluorescentprotein from Discosoma sp. (Matz, M et. al Nat Biotechnol 1999 Dec;17(12): 1227). Pyruvate kinase 332 3349-3680 Arabidopsis thalianapyruvate terminator kinase terminator (At5g52920) Bacterial Kanamycin817 3825-4641 Bacterial kanamycin selectable marker Act2 terminator 8004823-5622 Arabidopsis thaliana Actin 2 terminator NptII 795 5685-6479Neomycin phosphotransferase II plant selectable marker UBQ10 intron 3596507-6865 PCR amplified Arabidopsis thaliana intron from UBQ10 gene(At4g05320) for stabilization of NptII gene transcript and increaseprotein expression levels TEF2 Promoter 2000 6880-8879 Saccharomycescerevisiae translation elongation factor alpha promoter for expressionof NptII LoxP 34 312-345 & 8898-8931 Recombination site for Cre mediatedrecombination (Arenski et. al 1983, Abremski et. al 1984)

In the pCHR488 mini-chromosome donor vector, the yeast TEF2 promoter ofpCHR487 was replaced with the yeast GPD1 promoter which drove the plantselectable marker NptII. The yeast GPD1 promoter was PCR amplified fromSaccharomyces cerevisiae genomic DNA using standard PCR methods.Standard cloning methods were also used to replace the TEF2 promoter andinsert the yeast GPD1 promoter. For construction of mini-chromosomes,donor pCHR488 was generated as described for pCHR151. As with pCHR151,the circular donor pCHR488 lacks a bacterial origin of replication andthe bacterial ampicillin selectable marker. The donor pCHR488 constructwas used to construct plant mini-chromosomes as described for pCHR151.Mini-chromosome genetic elements within the pCHR488 vector are set outin Table 13 TABLE 13 Donor Components of pCHR488 Size Genetic Element(base pair) Location (bp) Details UBQ10 promoter 2038  361-2398Arabidopsis thaliana polyubiquitin promoter (At4g05320) DsRed2 + NLS 7802435-3214 Nuclear localized red fluorescent protein from Discosoma sp.(Matz, M et. al Nat Biotechnol 1999 Dec; 17(12): 1227). Pyruvate kinase332 3349-3680 Arabidopsis thaliana pyruvate terminator kinase terminator(At5g52920) Bacterial Kanamycin 817 3825-4641 Bacterial kanamycinselectable marker Act2 terminator 800 4823-5622 Arabidopsis thalianaActin 2 terminator NptII 795 5685-6479 Neomycin phosphotransferase IIplant selectable marker UBQ10 intron 359 6500-6859 PCR amplifiedArabidopsis thaliana intron from UBQ10 gene (At4g05320) forstabilization of NptII gene transcript and increase protein expressionlevels GPD1 Promoter 2000 6880-8879 Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase (NAD+) promoter for expression of NptII LoxP34 312-345 & 8898-8931 Recombination site for Cre mediated recombination(Arenski et. al 1983, Abremski et. al 1984)

In the pCHR489 mini-chromosome donor vector, the yeast TEF2 promoter ofpCHR487 was replaced with the Drosophila melanogaster Grim fly promoterfor driving the plant selectable marker NptII. The Grim fly promoter wasPCR amplified from Drosophila melanogaster genomic DNA using standardPCR methods. Standard cloning methods were used to replace the TEF2promoter in pCHR487 with the Grim fly promoter to generate pCHR489. Forconstruction of mini-chromosomes, donor pCHR489 was generated asdescribed for pCHR151. As with pCHR151, the circular donor pCHR489 lacksa bacterial origin of replication and the bacterial ampicillinselectable marker. The donor pCHR489 construct was used to constructplant mini-chromosomes as described for pCHR151. Mini-chromosome geneticelements within the pCHR489 vector are set out in Table 14. TABLE 14Donor Components of pCHR489 Size Genetic Element (base pair) Location(bp) Details UBQ10 promoter 2038  361-2398 Arabidopsis thalianapolyubiquitin promoter (At4g05320) DsRed2 + NLS 780 2435-3214 Nuclearlocalized red fluorescent protein from Discosoma sp. (Matz, M et. al NatBiotechnol 1999 Dec; 17(12): 1227). Pyruvate kinase 332 3349-3680Arabidopsis thaliana pyruvate terminator kinase terminator (At5g52920)Bacterial Kanamycin 817 3825-4641 Bacterial kanamycin selectable markerAct2 terminator 800 4823-5622 Arabidopsis thaliana Actin 2 terminatorNptII 795 5685-6479 Neomycin phosphotransferase II plant selectablemarker UBQ10 intron 359 6507-6865 PCR amplified Arabidopsis thalianaintron from UBQ10 gene (At4g05320) for stabilization of NptII genetranscript and increase protein expression levels Grim Fly Promoter 21916880-8879 PCR amplified promoter of grim (AKA BcDNA: RE28551) fromDrosophila melanogaster LoxP 34 312-345 & 9081-9114 Recombination sitefor Cre mediated recombination (Arenski et. al 1983, Abremski et. al1984)Vectors Used to Construct Mini-Chromosomes Via Standard Cloning Methods:

pCHR510

As in pCHR151, pCHR510 contains DsRed with a nuclear localization signaland is regulated by the Arabidopsis UBQ10 promoter. The Arabidopsispyruvate kinase terminator (At5g52920) was replaced by standard cloningprocedures with the Arabidopsis thaliana triose phosphate isomeraseterminator to prevent redundant use of the Arabidopsis pyruvate kinaseterminator (At5g52920) in pCHR510. In addition, the E. coli MerA genecassette was replaced with the plant selectable marker NptII regulatedby the Drosophila melanogaster Grim fly promoter plus Arabidopsis UBQ10intron and the Arabidopsis pyruvate kinase terminator (At5g52920). Thevector also included a ZsGreen fluorescent gene (Clontech Corporation,Palo Alto Calif.) regulated by the Arabidopsis Act2 promoter plusnaturally occurring intron and the Arabidopsis Act2 terminator. Thehigh-copy E. coli backbone of pUC19 and ampicillin bacterial selectablemarker were replaced with the low copy pBeloBAC11 backbone with thebacterial streptomycin resistance gene replacing the chloramphenicolresistance gene. An Arabidopsis thaliana ST11 sub-telomeric fragment wasintroduced upstream of the Grim fly promoter to isolate the Grim flypromoter from possible promoter silencing when a centromere fragment wasligated into the donor vector. Mini-chromosome genetic elements withinthe pCHR510 vector are set out in Table 15 below. TABLE 15 pCHR510 DNAdonor components Size (base Genetic Element pairs) Location (bp) DetailsBacterial 10111 16912-17922 Bacterial selectable marker streptomycinresistance ori2 67 19158-19224 F′ plasmid origin of replication from E.coli repE 755 19553-20308 mediation of replication complex at Ori2(Mori, H et. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15) SopA 116620896-22062 partition of plasmid to bacterial daughter cells (Mori, Het. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15) SopB 971 22062-23033partition of plasmid to bacterial daughter cells (Mori, H et. al, J MolBiol. 1986 Nov 5; 192(1): 1-15) SopC 517 23106-23623 partition ofplasmid to bacterial daughter cells (Mori, H et. al, J Mol Biol. 1986Nov 5; 192(1): 1-15) LoxP 34 26-59 and Recombination site for Cre16212-16245 mediated recombination (Arenski et. al 1983, Abremski et. al1984) ST11 subtelomeric 4682  69-4750 Arabidopsis thaliana DNA(complementary) subtelomenic DNA from Chromosome 5 Grim Promoter 21874766-6956 PCR amplified Drosophila melanogaster Grim gene promoter forexpression of NptII gene in plants. UBQ10 intron 359 6963-7322 PCRamplified Arabidopsis thaliana intron from UBQ10 gene (At4g05320) forstabilization of NptII gene transcript and increase protein expressionlevels. NptII 795 7350-8144 Neomycin phosphotransferase II plantselectable marker Pyruvate kinase 332 8212-8543 Arabidopsis thalianaPyruvate terminator kinase terminator (At5g52920) Bacterial 8178731-9547 Bacterial kanamycin selectable Kanamycin marker Act2promoter + intron 1482  9690-11171 The Arabidopsis thaliana promoterActin 2 plus natural intron. ZsGreen 695 11195-11890 Matz et. al. NatureBiotechnol. 1999 Oct; 17: 969 Act2 terminator 800 11931-12730Arabidopsis thaliana Actin2 gene terminator. Triose phosphate 45012759-13208 Arabidopsis thaliana Triose isomerase (complementary)phosphate isomerase gene terminator DsRed2 + NLS 780 13343-14122 Nuclearlocalized red (complementary) fluorescent protein from Discosoma sp.(Matz, M et. al Nat Biotechnol 1999 Dec; 17(12): 1227). UBQ10 Promoter2038 14159-16196 Arabidopsis thaliana (complementary) polyubiquitinpromoter (At4g05320)

To construct mini-chromosomes using pCHR510, the vector was linearizedusing standard restriction digestion procedures. The Brassica centromerefragment from mini-chromosome BB5R4-1 was restriction digested using NotI and ligated into pCHR510 using standard cloning procedures to generatethe mini-chromosome pCHR543. Mini-chromosomes were delivered into E.coli and grown in selective medium. Mini-chromosomes were extracted frombacteria and restriction digested to verify DNA composition and verifycentromere insert size.

pCHR579

The pCHR579 mini-chromosome donor vector was constructed using the samemethod to construct the pCHR510, without replacing the bacterialchloramphenicol gene in the low copy pBeloBAC11 backbone. Using standardcloning methods the bacterial kanamycin gene was replaced with abacterial kanamycin selectable marker surrounded by two plant telomeresequences and two unique I-PpoI homing endonuclease sequences asdescribed in pCHR171A. Mini-chromosomes using pCHR579 were constructedas described for pCHR510 using BB5R4-1 centromeric DNA to constructpCHR591. pCHR591 was linearized as described for mini-chromosomesdescribed above for pCHR171A. Mini-chromosome genetic elements withinthe pCHR579 vector are set out in Table 16 below. TABLE 16 pCHR579 DNAdonor components Size (base Genetic Element pairs) Location (bp) DetailsBacterial 660 18022-18681 Bacterial selectable chloramphenicol markerresistance ori2 67 19685-19751 F factor origin of replication from E.coli repE 755 20080-20835 mediation of replication complex at Ori2(Mori, H et. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15) SopA 1166214230-22589 partition of plasmid to bacterial daughter cells (Mori, Het. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15) SopB 971 22589-23560partition of plasmid to bacterial daughter cells (Mori, H et. al, J MolBiol. 1986 Nov 5; 192(1): 1-15) SopC 517 23633-24150 partition ofplasmid to bacterial daughter cells (Mori, H et. al, J Mol Biol. 1986Nov 5; 192(1): 1-15) LoxP 34 26-59 Recombination site for Cre mediatedrecombination (Arenski et. al 1983, Abremski et. al 1984) ST11sub-telomeric 4682  69-4750 Arabidopsis thaliana DNA (complementary)subtelomeric DNA from Chromosome 5 Grim Fly Promoter 2187 4766-6956 PCRamplified Drosophila melanogaster Grim gene promoter for expression ofNptII gene in plants. UBQ10 intron 359 6963-7322 PCR amplifiedArabidopsis thaliana intron from UBQ10 promoter (At4g05320) forstabilization of NptII gene transcript and increase protein expressionlevels. NptII 795 7350-8144 Kanamycin plant selectable marker Pyruvatekinase 332 8212-8543 Arabidopsis thaliana terminator Pyruvate kinaseterminator (At5g52920) Plant telomere 759 8598-9356 Plant telomere PCRbased on plant consensus telomere sequence Bacterial 817  9532-10348Bacterial kanamycin Kanamycin selectable marker Plant telomere 75910482-11241 Plant telomere PCR based on plant consensus telomeresequence Act2 promoter + 1482 11287-12768 The Arabidopsis thalianaintron promoter Actin 2 plus natural intron. ZsGreen 695 12792-13487Matz et.al. Nature Biotechnol. 1999 Oct; 17: 969 Act2 terminator 80013528-14327 Arabidopsis thaliana Actin2 gene terminator. Triosephosphate 450 14356-14805 Arabidopsis thaliana isomerase (complementary)Triose phosphate isomerase gene terminator DsRed2 + NLS 780 14940-15719Nuclear localized red (complementary) fluorescent protein from Discosomasp. (Matz, M et. al Nat Biotechnol 1999 Dec; 17(12): 1227). UBQ10Promoter 2038 15756-17793 Arabidopsis thaliana (complementary)polyubiquitin promoter (At4g05320)

pCHR581

The pCHR581 mini-chromosome donor vector was constructed using standardcloning procedures. The vector was constructed as pCHR579 with no ST11sub-telomeric DNA. Mini-chromosome genetic elements within the pCHR581vector are set out in Table 17 below.

ST9 is an Arabidopsis thaliana sub-telomeric sequence from centromere 5(bases 3708-195 (3513 bp); Database: ATH1_chr5.1con) , which wasamplified with the following oligo nucleotides: CHHZ-199(GGTGGTCGGCCGGAGCACAA GCGGGCCAAGCCCATGCTTG; SEQ ID NO: 29) and CHHZ-202(GGTGGTCGGCCGCAGGTTGCATATGAATCTTTA ACTGACAG; SEQ ID NO: 30). ST1 is anArabidopsis thaliana sub-telomeric sequence from centromere 5 (bases195-3708 (3513 bp); Database: ATH1_chr5.1con), which was amplified withthe following oligo nucleotides: CHHZ-200(GGTGGTCGGCCGCGAGCACAAGCGGGCCAAGCCCATGCTTG; SEQ ID NO: 31) and CHHZ-201(GGTGGTCGGCCGTCAGGTTGCATATGAATCTT TAACTGACAG: SEQ ID NO: 32). ST11 is anArabidopsis thaliana sub-telomeric sequence from centromere 5 (bases26,987,774-26,992,453 (4681 bp); Database: ATH1_chr5.1 con), which wasamplified with the following oligo nucleotides: CHHZ-203(GGTGGTCGGCCGTCGTCGGCACTTGGCAGCGAAATCTCC; SEQ ID NO: 33) and CHHZ-206(GGTGGTCGGCCGCATTATCATATAATTATGTTT TGCTGCTTC: SEQ ID NO: 34). ST12 is anArabidopsis thaliana sub-telomeric sequence from centromere 5 (bases26,992,453-26,987,774 (4681 bp); Database: ATH1_chr5.1con), which wasamplified with the following oligo nucleotides: CHHZ-204(GGTGGTCGGCCGCGTCGGCACTTGGCAGCGAAATCTCC; SEQ ID NO: 35) and CHHZ-205(GGTGGTCGGCCGATTATCATATAATTATGT TTTGCTGCTTC: SEQ ID NO: 36). Thesesub-telomeric sequences were included in the pCRR581 vector. TABLE 17pCHR581 DNA donor components Size (base Genetic Element pairs) Location(bp) Details Bacterial 660 13333-13992 Bacterial selectable markerchloramphenicol resistance ori2 67 14996-15062 F′ plasmid origin ofreplication from E. coli repE 755 15391-16146 mediation of replicationcomplex at Ori2 (Mori, H et. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15)SopA 1166 16734-17900 partition of plasmid to bacterial daughter cells(Mori, H et. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15) SopB 97117900-18871 partition of plasmid to bacterial daughter cells (Mori, Het. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15) SopC 517 18944-19461partition of plasmid to bacterial daughter cells (Mori, H et. al, J MolBiol. 1986 Nov 5; 192(1): 1-15) LoxP 34 26-59 Recombination site for Cremediated recombination (Arenski et. al 1983, Abremski et. al 1984) GrimPromoter 2187  77-2267 PCR amplified Drosophila melanogaster Grim genepromoter for expression of NptII gene in plants UBQ10 intron 3592274-2633 PCR amplified Arabidopsis thaliana intron from UBQ10 gene(At4g05320) for transcript and increase protein expression levels. NptII795 2661-3455 Neomycin phosphotransferase II plant selectable markerPyruvate kinase 332 3523-3854 Arabidopsis thaliana Pyruvate terminatorkinase terminator (At5g52920) Plant telomere 759 3909-4667 Planttelomere PCR based on plant consensus telomere sequence BacterialKanamycin 817 4843-5659 Bacterial kanamycin selectable marker Planttelomere 759 5793-6552 Plant telomere PCR based on plant consensustelomere sequence Act2 promoter + intron 1482 6598-8079 The Arabidopsisthaliana promoter Actin 2 plus natural intron. ZsGreen 695 8103-8798Matz et. al. Nature Biotechnol. 1999 Oct; 17: 969 Act2 terminator 8008839-9638 Arabidopsis thaliana Actin2 gene terminator. Triose phosphate450  9667-10116 Arabidopsis thaliana Triose isomerase (complementary)phosphate isomerase gene terminator DsRed2 + NLS 780 10251-11030 Nuclearlocalized red (complementary) fluorescent protein from Discosoma sp.(Matz, M et. al Nat Biotechnol 1999 Dec; 17(12): 1227). UBQ10 Promoter2038 11067-13104 Arabidopsis thaliana (complementary) polyubiquitinpromoter (At4g05320)

pCHR806

The pCHR806 mini-chromosome vector was constructed using standardcloning procedures. The vector was composed similarly to that of pCHR510using the pBeloBAC11 low copy backbone containing the bacterialchloramphenicol gene and without the addition of the ST11 sub-telomericDNA. An additional plant gene cassette was introduced containing theAnemonia sp. cyan fluorescence (AmCyan) gene regulated by the tomatoLat52 promoter and terminator. Mini-chromosome genetic elements withinthe pCHR806 vector are set out in Table 18. TABLE 18 pCHR806 GeneticElement Size (base pairs) Location (bp) Details Bacterial 66013372-14031 Bacterial selectable marker chloramphenicol resistance ori267 15035-15101 F′ plasmid origin of replication from E. coli repE 75515430-16185 mediation of replication complex at Ori2 (Mori, H et. al, JMol Biol. 1986 Nov 5; 192(1): 1-15) SopA 1166 16773-17939 partition ofplasmid to bacterial daughter cells (Mori, H et. al, J Mol Biol. 1986Nov 5; 192(1): 1-15) SopB 971 17939-18910 partition of plasmid tobacterial daughter cells (Mori, H et. al, J Mol Biol. 1986 Nov 5;192(1): 1-15) SopC 517 18983-19500 partition of plasmid to bacterialdaughter cells (Mori, H et. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15)LoxP 34 26-59 Recombination site for Cre mediated recombination (Arenskiet. al 1983, Abremski et. al 1984) Grim Promoter 2187  77-2267 PCRamplified Drosophila melanogaster Grim gene promoter for expression ofNptII gene in plants. UBQ10 intron 359 2274-2633 PCR amplifiedArabidopsis thaliana intron from UBQ10 gene (At4g05320) forstabilization of NptII gene transcript and increase protein expressionlevels. NptII 795 2661-3455 Neomycin phosphotransferase II plantselectable marker Pyruvate kinase 332 Arabidopsis thaliana terminatorPyruvate kinase terminator (At5g52920) Lat52 terminator 200 3883-4082Tomato Lat52 terminator (complementary) AmCyan 690 4123-4812 Visiblecyan fluorescent (complementary) protein from Anemonia majano (Matz, Met. al Nat Biotechnol 1999 Dec; 17(12): 1227). Tomato Lat52 6684836-5503 Tomato Lat52 promoter promoter (complementary) BacterialKanamycin 817 5678-6494 Bacterial kanamycin selectable marker Act2promoter + intron 1482 6637-8118 The Arabidopsis thaliana promoter Actin2 plus natural intron. ZsGreen 695 8142-8837 Matz et. al. NatureBiotechnol. 1999 Oct; 17: 969 Act2 terminator 800 8878-9677 Arabidopsisthaliana Actin2 gene terminator. Triose phosphate 450  9706-10155Arabidopsis thaliana Triose isomerase phosphate isomerase geneterminator DsRed2 + NLS 780 10290-11069 Nuclear localized red(complementary) fluorescent protein from Discosoma sp. (Matz, M et. alNat Biotechnol 1999 Dec; 17(12): 1227). UBQ10 Promoter 2038 11106-13143Arabidopsis thaliana (complementary) polyubiquitin promoter (At4g05320)

pCHR807

The pCHR807 mini-chromosome donor vector was constructed using standardcloning procedures and is identical to pCHR806. The vector was composedsimilar to that of pCHR510 using the pBelloBAC11 low copy backbonecontaining the bacterial chloramphenicol gene and without the additionof the ST11 sub-telomeric DNA. An additional plant gene cassette wasintroduced containing the Zoanthus sp. yellow fluorescent gene(ZsYellow) regulated by the tomato Lat52 promoter and terminator.Mini-chromosome genetic elements within the pCHR807 vector are set outin Table 19. TABLE 19 pCHR807 DNA donor components Size (base GeneticElement pairs) Location (bp) Details Bacterial 660 13378-14037 Bacterialselectable marker chloramphenicol resistance ori2 67 15041-15107 F′plasmid origin of replication from E. coli repE 755 15436-16191mediation of replication complex at Ori2 (Mori, H et. al, J Mol Biol.1986 Nov 5; 192(1): 1-15) SopA 1166 16779-17945 partition of plasmid tobacterial daughter cells (Mori, H et. al, J Mol Biol. 1986 Nov 5;192(1): 1-15) SopB 971 17945-18916 partition of plasmid to bacterialdaughter cells (Mori, H et. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15)SopC 517 18989-19506 partition of plasmid to bacterial daughter cells(Mori, H et. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15) LoxP 34 26-59Recombination site for Cre mediated recombination (Arenski et. al 1983,Abremski et. al 1984) Grim Promoter 2187  77-2267 PCR amplifiedDrosophila melanogaster Grim gene promoter for expression of NptII genein plants. UBQ10 intron 359 2274-2633 PCR amplified Arabidopsis thalianaintron from UBQ10 gene (At4g05320) for stabilization of NptII genetranscript and increase protein expression levels. NptII 795 2661-3455Neomycin phosphotransferase II plant selectable marker Pyruvate kinase332 3523-2854 Arabidopsis thaliana Pyruvate terminator kinase terminator(At5g52920) Lat52 terminator 200 3883-4082 Tomato Lat52 terminator(complementary) ZsYellow 696 4123-4818 Visible yellow fluorescent(complementary) protein from Zoanthus sp. (Matz, M et. al Nat Biotechnol1999 Dec; 17(12): 1227). Tomato Lat52 668 4842-5509 Tomato Lat52promoter promoter Bacterial 817 5684-6500 Bacterial kanamycin Kanamycinselectable marker Act2 promoter + intron 1482 6643-8124 The Arabidopsisthaliana promoter Actin 2 plus natural intron. ZsGreen 695 8148-8843Matz et. al. Nature Biotechnol. 1999 Oct; 17: 969 Act2 terminator 8008884-9683 Arabidopsis thaliana Actin2 gene terminator. Triose phosphate450  9712-10161 Arabidopsis thaliana Triose isomerase phosphateisomerase gene terminator DsRed2 + NLS 780 10296-11075 Nuclear localizedred fluorescent protein from Discosoma sp. (Matz, M et. al NatBiotechnol 1999 Dec; 17(12): 1227). UBQ10 Promoter 2038 11112-13149Arabidopsis thaliana polyubiquitin promoter (At4g05320)

pCHR808

The pCHR808 mini-chromosome donor vector was constructed using standardcloning procedures and is identical to pCHR806, but with the addition ofthe Arabidopsis thaliana ST9 sub-telomeric DNA. The ST9 sub-telomericfragment was introduced upstream of the Grim fly promoter to isolate theGrim fly promoter from possible promoter silencing when a centromerefragment is ligated into the donor vector. Mini-chromosome geneticelements within the pCHR808 vector are set out in Table 20. TABLE 20pCHR808 DNA donor components Size (base Genetic Element pairs) Location(bp) Details Bacterial 660 16892-17551 Bacterial selectablechloramphenicol marker resistance ori2 67 18555-18621 F′ plasmid originof replication from E. coli repE 755 18950-19705 mediation ofreplication complex at Ori2 (Mori, H et. al, J Mol Biol. 1986 Nov 5;192(1): 1-15) SopA 1166 20293-21459 partition of plasmid to bacterialdaughter cells (Mori, H et. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15)SopB 971 21459-22430 partition of plasmid to bacterial daughter cells(Mori, H et. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15) SopC 51722503-23020 partition of plasmid to bacterial daughter cells (Mori, Het. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15) LoxP 34 26-59Recombination site for Cre mediated recombination (Arenski et. al 1983,Abremski et. al 1984) ST9 subtelomeric 3513  69-3581 Arabidopsisthaliana DNA subtelomeric DNA from Chromosome 5 Grim Promoter 21873597-5787 PCR amplified Drosophila melanogaster Grim gene promoter forexpression of NptII gene in plants. UBQ10 intron 359 5794-6153 PCRamplified Arabidopsis thaliana intron from UBQ10 gene (At4g05320) forstabilization of NptII gene transcript and increase protein expressionlevels. NptII 795 6181-6975 Neomycin phosphotransferase II plantselectable marker Pyruvate kinase 332 7043-7374 Arabidopsis thalianaterminator Pyruvate kinase terminator (At5g52920) Lat52 terminator 2007403-7602 Tomato Lat52 terminator (complementary) AmCyan 690 7643-8332Visible cyan fluorescent protein from Anemonia majano (Matz, M et. alNat Biotechnol 1999 Dec; 17(12): 1227). Tomato Lat52 668 8356-9023Tomato Lat52 promoter promoter (complementary) Bacterial 817  9198-10014Bacterial kanamycin Kanamycin selectable marker Act2 promoter + 148210157-11638 The Arabidopsis thaliana intron promoter Actin 2 plusnatural intron. ZsGreen 695 11662-12357 Matz et.al. Nature Biotechnol.1999 Oct; 17: 969 Act2 terminator 800 12398-13197 Arabidopsis thalianaActin2 gene terminator. Triose phosphate 450 13226-13675 Arabidopsisthaliana isomerase Triose phosphate isomerase gene terminator DsRed2 +NLS 780 13810-14589 Nuclear localized red fluorescent protein fromDiscosoma sp. (Matz, M et. al Nat Biotechnol 1999 Dec; 17(12): 1227).UBQ10 Promoter 2038 14626-16663 Arabidopsis thaliana polyubiquitinpromoter (At4g05320)

pCHR945

The pCHR945 mini-chromosome donor vector was constructed using standardcloning procedures and is identical to pCHR807 with the replacement ofthe bacterial kanamycin gene with a bacterial kanamycin selectablemarker gene surrounded by two plant telomere sequences and two uniqueI-Ppo I homing endonuclease sequences as described in pCHR171A.Mini-chromosomes using pCHR945 were constructed as described for pCHR510using BB5R4-1 centromeric DNA to construct pCHR955. pCHR955 waslinearized as described for mini-chromosomes constructed with pCHR171A.Mini-chromosome genetic elements within the pCHR845 vector are set outin Table 21. TABLE 21 pCHR945 DNA donor components Size (base GeneticElement pairs) Location (bp) Details Bacterial chloramphenicol 66014992-15651 Bacterial selectable marker resistance ori2 67 16655-16721F′ plasmid origin of replication from E. coli repE 755 17050-17805mediation of replication complex at Ori2 (Mori, H et. al, J Mol Biol.1986 Nov 5; 192(1): 1-15) SopA 1166 18393-19559 partition of plasmid tobacterial daughter cells (Mori, H et. al, J Mol Biol. 1986 Nov 5;192(1): 1-15) SopB 971 19559-20530 partition of plasmid to bacterialdaughter cells (Mori, H et. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15)SopC 517 20603-21120 partition of plasmid to bacterial daughter cells(Mori, H et. al, J Mol Biol. 1986 Nov 5; 192(1): 1-15) LoxP 34 26-59Recombination site for Cre mediated recombination (Arenski et. al 1983,Abremski et. al 1984) Grim Promoter 2187  77-2267 PCR amplifiedDrosophila melanogaster Grim gene promoter for expression of NptII genein plants. UBQ10 intron 359 2274-2633 PCR amplified Arabidopsis thalianaintron from UBQ10 gene (At4g05320) for stabilization of NptII genetranscript and increase protein expression levels. NptII 795 2661-3455Neomycin phosphotransferase II plant selectable marker Pyruvate kinaseterminator 332 3523-3854 Arabidopsis thaliana Pyruvate kinase terminator(At5g52920) Lat52 terminator 200 3883-4082 Tomato Lat52 terminator(complementary) ZsYellow 696 4123-4818 Visible yellow fluorescent(complementary) protein from Zoanthus sp. (Matz, M et. al Nat Biotechnol1999 Dec; 17(12): 1227). Tomato Lat52 promoter 668 4842-5509 TomatoLat52 promoter (complementary) Plant telomere 759 5568-6326 Planttelomere PCR based on plant consensus telomere sequence BacterialKanamycin 817 6502-7318 Bacterial kanamycin selectable marker Planttelomere 759 7452-8211 Plant telomere PCR based on plant consensustelomere sequence Act2 promoter + intron 1482 8257-9738 The Arabidopsisthaliana promoter Actin 2 plus natural intron. ZsGreen 695  9762-10457Matz et.al. Nature Biotechnol. 1999 Oct; 17: 969 Act2 terminator 80010498-11297 Arabidopsis thaliana Actin2 gene terminator. Triosephosphate isomerase 450 11326-11775 Arabidopsis thaliana Trioseterminator (complementary) phosphate isomerase gene terminator DsRed2 +NLS 780 11910-12689 Nuclear localized red (complementary) fluorescentprotein from Discosoma sp. (Matz, M et. al Nat Biotechnol 1999 Dec;17(12): 1227). UBQ10 Promoter 2038 12726-14763 Arabidopsis thaliana(complementary) polyubiquitin promoter (At4g05320)

Other Vectors

The pCHR809 mini-chromosome donor vector was constructed using standardcloning procedures and is identical to pCHR807, but with the addition ofthe Arabidopsis thaliana ST9 sub-telomeric DNA. The ST9 sub-telomericfragment was introduced upstream of the Grim fly promoter to isolate theGrim fly promoter from possible promoter silencing when a centromerefragment was ligated into the donor vector.

The pCHR810 mini-chromosome donor vector was constructed using standardcloning procedures and is identical to pCHR806, but with the addition ofthe Arabidopsis thaliana ST10 sub-telomeric DNA. The ST10 sub-telomericfragment was introduced upstream of the Grim fly promoter to isolate theGrim fly promoter from possible promoter silencing when a centromerefragment was ligated into the donor vector.

The pCHR811 mini-chromosome donor vector was constructed using standardcloning procedures and is identical to pCHR807, but with the addition ofthe Arabidopsis thaliana ST10 sub-telomeric DNA. The ST10 sub-telomericfragment was introduced upstream of the Grim fly promoter to isolate theGrim fly promoter from possible promoter silencing when a centromerefragment was ligated into the donor vector.

The pCHR813 mini-chromosome donor vector was constructed using standardcloning procedures and is identical to pCHR807, but with the addition ofthe Arabidopsis thaliana ST11 sub-telomeric DNA. The ST11 sub-telomericfragment was introduced upstream of the Grim fly promoter to isolate theGrim fly promoter from possible promoter silencing when a centromerefragment was ligated into the donor vector.

The pCHR814 mini-chromosome donor vector was constructed using standardcloning procedures and is identical to pCHR806, but with the addition ofthe Arabidopsis thaliana ST11 sub-telomeric DNA. The ST12 sub-telomericfragment was introduced upstream of the Grim fly promoter to isolate theGrim fly promoter from possible promoter silencing when a centromerefragment was ligated into the donor vector.

The pCHR815 mini-chromosome donor vector was constructed using standardcloning procedures and is identical to pCHR807, but with the addition ofthe Arabidopsis thaliana ST11 sub-telomeric DNA. The ST12 sub-telomericfragment was introduced upstream of the Grim fly promoter to isolate theGrim fly promoter from possible promoter silencing when a centromerefragment was ligated into the donor vector.

The pCHR948 mini-chromosome donor vector was constructed using standardcloning procedures and is identical to pCHR810 with the replacement ofthe bacterial kanamycin gene with a bacterial kanamycin selectablemarker gene surrounded by two plant telomere sequences and two uniqueI-Ppo I homing endonuclease sequences as described in pCHR171A.Mini-chromosomes using pCHR948 were constructed as described for pCHR510using BB5R4-1 centromeric DNA. pCHR958 was linearized as described formini-chromosomes constructed with pCHR171A.

EXAMPLE 3 Mini-Chromosome Delivery into Brassica Cells

Various methods may be used to deliver DNA into plant cells. Theseinclude biological methods, such as Agrobacterium and viruses, physicalmethods such as biolistic particle bombardment and silicon carbidewhiskers, electrical methods such as electroporation, and chemicalmethods such as the use of poly-ethylene glycol and other compoundsknown to stimulate DNA uptake into cells. Agrobacterium and biolisticparticle bombardment have been the methods that have found mostwidespread use in plant biotechnology. See, e.g., Broothaerts, et. al.Nature 433: 629-633, 2005.

Biolistic Particle Delivery of Mini-Chromosomes

A biolistic delivery method using wet gold particles kept in an aqueousDNA suspension was adapted from the teachings of Milahe and Miller(Biotechniques 16: 924-931, 1994) and used to transform B. oleracea(Broccoli) cells. To prepare the wet gold particles for bombardment, 1.0μm gold particles were washed by mixing with 100% ethanol on a vortexfollowed by spinning the particles in a microfuge at 4000 rpm in orderto remove supernatant. Subsequently, the gold particles were washed withsterile distilled water three times, followed by spinning in a microfugeto remove supernatant. The washed gold particles were resuspend insterile distilled water at a final concentration of 90 mg/ml and storedat 4° C. until use. For bombardment, the gold particle suspension (90mg/ml)was then mixed rapidly with 1 μg/μl DNA solution (in dH₂O or TE),2.5M CaCl₂, and 1M spermidine. When two or more plasmids were containedwithin the DNA solution, equal amounts of each plasmids was added to thegold suspension.

To prepare explant tissues for DNA delivery, three days prior tobombardment, an internode of the Brassica plant (Broccoli) were cut. Theinternode explant was cut longitudinally with a scalpel to cut a thinslice (⅙-¼ of the internode) off one side of the explant. The preparedinternodes were placed wound side down on Petri dishes with regenerationmedia. The Petri dishes were wrapped with tape and placed wound side upunder the light. The explants grew for 3 days prior to bombardment.

For bombardment of Brassica suspension cells, the cells were harvestedby centrifugation (1200 rpm for 2 minutes) on the day of bombardment.The cells were plated onto 50 mm circular polyester screen cloth disksplaced on petri plates with solid medium. The solid medium used was thesame medium that the cells are normally grown in (MS salts, Gamborg'svitamins, 3% sucrose, 2 mg/liter 2,4D (2,4-Dichlorophenoxyacetic acid),0.5 mM MES pH 5.8+(solid medium only), plus 0.26% gelrite, or 0.6%tissue culture agar, added before autoclaving. Approximately 1.5 mlpacked cells were placed on each filter disk, and dispersed uniformlyinto a very even spot approximately 1 inch in diameter.

Bombardment of the cells was carried out in the BioRad PDS-1000/HeBiolistic Particle Delivery System (BioRad). The DNA/gold suspension wasresuspended and immediately inserted onto the grid of the filter holder.A 50 mm circular polyester screen cloth disk containing the cells wasplaced into a fresh 60 mm petri dish and the cells were covered with a10×10 cm square of sterile nylon or Dacron chiffon netting. The metalcylinder was inserted into the petri dish and used to push the nettingdown to the bottom of the dish. This weight prevented the cells frombeing dislodged from the plate during bombardment. The petri dishcontaining the cells was then placed onto the sample holder, andpositioned in the sample chamber of the gene gun and bombarded with theDNA/gold suspension. After the bombardment, the cells were scraped offthe filter circle in the petri dish containing solid medium with asterile spatula and transferred to fresh medium in a 125 ml blue-cappedglass bottle. The bottles were transferred onto a shaker and grown whileshaking at 150 rpm.

A biolistic delivery method using dry gold particles was also carriedout to deliver mini-chromosomes to Brassica cells. For this method, 1.0or 0.6μ gold particles were washed in 70% ethanol with vigorous shakingon a vortex for 3 to 5 minutes, followed by an soaking in 70% ethanolfor 15 minutes. The gold particles were spun in a microfuge to removethe supernatant and washed three times in sterile distilled water. Thegold particles were suspended in 50% glycerol at a concentration of 60mg/ml and stored at 4° C. For bombardment, the dry gold particles wereresuspended on a vortex for 5 minutes to disrupt agglomerated particles.Subsequently, the dry gold particles were mixed rapidly with DNA, 2.5MCaCl₂ and 0.2M spermidine in a siliconized, sterile eppendorf tube. Thesample was allowed to settle for 1 minute and then spun in a microfugefor 10 seconds to remove supernatant. Subsequently, the DNA/goldparticles were washed once with 70% ethanol, followed by two washed in100% ethanol. A portion of the DNA/gold mixture was evenly placed on amacrocarrier. The macrocarrier was then placed in the BioRad PDS-1000/HeBiolistic Particle Delivery System, and the bombardment was done atrupture disk pressures ranging from 450 psi to 2,200 psi. The drybiolistic method did not result in the generation of adchromosomalplants or cell lines.

EXAMPLE 4 Selection of Brassica Cell Clones Stably ContainingMini-Chromosome DNA

Use of Visible Marker Genes

The presence of visible marker genes allowed for visual selection ofBrassica cells stably containing mini-chromosome DNA because anyadchromosomal cells or cell clusters were readily identified by virtueof fluorescent protein expression.

Transient assays were used to test mini-chromosomes for their ability tobecome established in cells following DNA delivery, and for theirability to be inherited in mitotic cell divisions. Expression of avisible marker encoded by a gene present on the mini-chromosome, such asa fluorescent protein gene, is used to detect mini-chromosome presencein the cell, and to follow mitotic inheritance of the mini-chromosome.In this assay, mini-chromosomes were delivered to Brassica cells of apopulation that is undergoing cell division, in this case Brassicasuspension cells grown in liquid culture.

After DNA delivery, the cell population was monitored for fluorescentprotein expression over the course of one to several weeks.Mini-chromosomes containing active centromeres were observed through theformation of fluorescent cell clusters, which were derived from a singleprogenitor cell that had divide and passed the mini-chromosomes to itsdaughter cells. Accordingly, single fluorescent cells and clusters offluorescent cells of various sizes were scored in the growing cellpopulation after DNA delivery. A total of 25 Brassica mini-chromosomes(see Table 23), constructed using the cre-lox assembly process, weretested in this manner in several different Brassica cell lines. A numberof mini-chromosomes showed indications of stable mitotic inheritance inthis assay and are listed in Table 22; in addition several stable celllines were obtained from suspension cell lines following the delivery ofthe same mini-chromosomes; these are also listed in Table 22. TABLE 22Preferred Brassica BACs, centromeres (CEN), and mini-chromosomes (MC)based upon transient expression assays and generation of stable Brassicacell lines # of times # of Stable tested in positive BAC CEN MC clonestransient transient Number Number Number BAC Class generated deliveriesdeliveries BB5 BB5R4-1 BB5R4-1 Hi CANREP, 7 3 2 Meth BB16 BB16R1-2BB16R1-2 Hi CANREP, 0 2 1 Meth (Hpa) BB16R1-3 BB16R1-3 1 1 1 BB18BB18R1-2 BB18R1-2 Hi CANREP, 1 1 1 Meth (Hpa) BB18R2-3 BB18R2-3 1 4 2BB38 BB38R1-3 BB38R1-3 Hi CANREP 0 4 3 only BB47 BB47R1-2 BB47R1-2 HiCANREP, 0 4 2 Meth (Hpa) BB60 BB60R1-1 BB60R1-1 Hi CANREP, 1 3 3 Meth(Hpa) BB63 BB63R1-1 BB63R1-1 Hi CANREP, 1 5 4 Meth (Hpa) BB70 BB70R1-3BB70R1-3 Hi CANREP, 0 4 2 Moderate Meth BB71 BB71R1-3 BB71R1-1 HiCANREP, 0 2 2 Meth (Sau) BB76 BB76R1-3 BB76R1-3 Hi CANREP, 0 5 4Moderate Meth BB104 BB104R1-2 BB104R1-2 Hi CANREP, 1 3 3 Moderate MethManipulation of Adchromosomal Tissue to Homogeneity

After identifying clusters of fluorescent cells in bombarded suspensioncell cultures, physical manipulations were carried out to allow for thepreferential expansion of cells harboring the delivered genes.Non-fluorescent tissue surrounding the fluorescent clusters was trimmedto avoid overgrowth of fluorescent cells by non-fluorescent ones, whileretaining a minimum tissue size capable of rapid growth. Thesemanipulations were performed under sterile conditions with the use of afluorescence stereomicroscope that allows for visualization of thefluorescent cells and cell clumps in the larger pieces of tissue. Inbetween the mechanical purification steps, the tissue was allowed growthon appropriate media, either in the presence or absence of selection.Over time, a pure population of fluorescent cells was obtained.

EXAMPLE 5 Regeneration of Brassica Plants from Adchromosomal Cell Clones

A total of 28 Brassica mini-chromosomes were used in stabletransformation to successfully regenerate transchromosomal broccoliplants that are listed in Table 23. These Brassica mini-chromosomesrepresent candidate Brassica centromere sequences for the delivery andtransmission of stable Brassica mini-chromosomes. B. oleracea plant(broccoli) regeneration was achieved by cultivating pieces of sterileplant tissue (explants) on medium containing plant growth activators(auxins, cytokinins and other compounds) that induce embryogenesis orshoot formation. Particularly, Brassica tissue proliferation was carriedout with a medium containing high cytokinin regeneration medium(Murashige and Skooge salts, MES, sucrose, gelrite, Gamborg vitamins,6-benzylaminopurine hydrochloride, non-essential amino acids,thidiazuron (TDZ) and silver nitrate, pH 5.7) through all culturingphases. TABLE 23 In Growth size size Room # of of # # PCR³ RT³ Western³Centromere MC cofig genes #explants⁵ cen MC events plants visual² #events # events # events BB15R4-1 964-4 O-O 4 77 60 80 1 1 √ 1 0 1BB15R4-1 965-1 O-O 4 93 55 80 1 2 √ 1 0 1 BB16R1-2 967-1 O-O 4 105 55 601 1 √ 0 0 1 BB221 BB221R2-1 O-O 2 78 59 70 1 2 √ 0 0 1 BB222 BB222R2-7O-O 2 102 49 60 1 2 √ 1 0 0 BB229 BB229R2-6 O-O 2 118 49 60 3 8 √ 2 0 1BB280 BB280R2-3 O-O 2 79 86 97 3 6 √ 2 0 3 BB5 BB5R10-1 I-I 2 337 48 651 1 √ 1 0 0 BB5 BB5R14-6 O-O 2 213 52 68 1 1 √ 1 0 0 BB5 BB5R15-4 O-O 2144 52 68 1 2 √ 1 0 0 BB5 BB5R16-6 O-O 2 152 50 66 2 6 √ 1 1 1 BB5R4-1543-6 O-O 3 101 50 74 2 4 √ 1 0 0 BB5R4-1 591-1 I-O 3 76 50 75 1 2 √ 1 00 BB5R4-1 591-1L I-I 3 207 50 73 2 4 √ 2 1 1 BB5R4-1 593-3L I-I 3 78 5069 1 4 √ 1 1 1 BB5R4-1 593-4L I-I 3 82 50 69 1 3 √ 1 1 1 BB5 BB5R4-1 O-O2 564 50 61 7 46 √ 7 6 6 BB5R4-1 816-2 O-O 4 107 50 70 2 5 √ 2 1 1BB5R4-1 817-1 O-O 4 143 50 70 2 16 √ 2 2 2 BB5R4-1 818-1 O-O 4 78 50 731 1 √ 1 0 0 BB5R4-1 819-1 O-O 4 117 50 73 4 14 √ 4 4 4 BB5R4-1 820-1 O-O4 406 50 73 2 3 √ 2 0 1 BB5R4-1 823-2 O-O 4 50 50 75 3 16 √ 3 0 3BB5R4-1 824-9 O-O 4 129 50 75 2 4 √ 2 2 2 BB5R4-1 825-1 O-O 4 168 50 751 1 √ 0 0 1 BB5R4-1 958-2L I-I 4 169 50 74 1 1 √ 1 0 1 BB60R1-1 972-5O-O 4 123 60 80 1 2 √ 1 0 1 BB71 BB71R1-1 O-O 2 162 24 35 1 3 √ 1 1 1integrative 489 O-O 2 306 0 11 1 1 √ 1 1 1 SB38R2-2 986-1 O-O 2 61 63 833 5 √ 3 0 3 167

After bombardment, explants were returned to the high cytokininregeneration medium with the wound side down on the plate. The explantswere transferred to selection medium (regeneration medium containing 150μM HgCl₂ or 100 mg/L of kanamycin) three days after bombardment withwound side up. The explants were visually screened under a fluorescentdissecting microscope for red fluorescent cluster formation 10 daysafter selection was started. In addition to facilitating the transientassays, the use of fluorescent protein expression allowed for the use ofsub-killing concentrations of selective agent during growth of planttissue on selective medium. This flexibility allowed for the use of awider range of antibiotic concentrations than possible in the absence ofa visible marker gene, without having to consider the amount ofbackground growth observed in wild type plant tissue. Fluorescent cellclusters could be visually identified after one to several weeks ofgrowth on selective media. Clusters with some unmodified surroundingexplant tissue were carved out and placed on medium containing 50 μMHgCl₂ or 50 mg/L of kanamycin.

The subcultures were continued on selection medium and the non-modifiedtissues were parsed out from the clusters every week for a month. Oncethe clusters were approximately 3 mm in size, they were cultured onregeneration medium without any Hg or kanamycin in order to induce shootregeneration. Subsequently, shoot primordia were transferred to seedgermination medium for enlargement and expansion. Once the shootselongated and developed about 2-4 leaves, the shoots with the leaveswere cut off for rooting in rooting medium (MS salts, sucrose, 0.7%tissue culture agar and non-essential amino acids, pH 5.7). Once theshoots developed good root systems, they were potted and transferred toa plant growth facility.

EXAMPLE 6 Regeneration of B. Napus (Canola) Plants Modified withMini-Chromosome DNA

The biolistic delivery method using dry gold particles, described inexample 3, was used to deliver mini-chromosomes to B. napus hypocotylsections for the purpose of regenerating modified B. napus (hereinafter“canola”) plants. The canola hypocotyls were modified withmini-chromosomes generated with Brassica centromere inserts described inExample 1 using the vectors described in Example 1.

Canola seeds were grown in germination medium (1× Murashige and Skoog(MS) salts, 1× micro-salts with Gamborg's B5 vitamins (B5), 2% sucrose,2 g/liter gelrite, pH 5.8) for one week. The resulting hypocotyls wereharvested and sliced longitudinally; the pieces were cultured on callusinduction medium (1× MS salts, 1× MS vitamins, 1 mg/liter2,4-dichlorophenoxyacetic acid (2,4D), 3% sucrose, 2 g/liter gelrite, pH5.8) for 7 days.

The hypocotyls were bombarded with dry gold particle/DNA suspensions asdescribed in Example 2 using 1300 psi rupture disks; the bombardedtissues were returned to callus induction medium for 3 days and thentransferred to callus induction medium containing 10 mg/ml of G418. Theywere kept on this medium with sub-culturing every 1-2 weeks for 4-6weeks. The subcultures were then visually analyzed for expression of thefluorescent protein as described in Example 3.

Cultures positive for expression of the fluorescent protein were furthersubcultured on callus induction medium containing 10 mg/ml of G418 for4-6 weeks. During this time, fluorescent tissue was selectively isolatedfrom non-fluorescent tissue by manipulation. Subsequently, the positivetissues were transferred to organogenesis induction media (MS salts, B5vitamins, 6-benzylaminopurine, zeatin, sucrose, g/l gelrite, pH 5.8)containing 10 mg/ml of G418) and were kept on this medium until shootsappeared. The shoots were grown in hormone-free media to promote normalshoot development.

Developing green shoots with a defined morphology were excised andincubated in shoot elongation medium, differing from organogenesismedium by having lower cytokinin concentrations. Most callus was removedfrom the developing shoots, which were subcultured in fresh shootelongation medium every 2-3 weeks. As the developing shoots becamenormal and exhibited apical dominance, they were transferred to rootingmedium containing indolebutyric acid; the remaining callus was removedalso at this time. The shoots were arranged to stand in the medium withan exposed apex. The roots began to appear in 1-3 weeks.

The rooted shoots were transferred to soil in which the basal portion ofthe plant was planted to soil to grow out. The shoot were gently removedfrom the agar and large chunks of agar were removed by rinsing gently intap water. The roots were placed in wet RediEarth or other suitablegrowth medium. Roots were covered with the growth medium and packedgently. The shoots were hardened-off and acclimated to growing in soilby covering the shoot with a clear container. The shoots were placedinto a greenhouse or plant growth room. After being covered for 3-4days, the shoots were gradually exposed to room air by partial removalof the cover. Once the plant stopped wilting, the cover was removedentirely.

Several adchromosomal events and approximately 30 adchromosomal plantswere obtained by this protocol; these are further discussed in example12 and table 45. To visually analyze the presence of the marker gene inBrassica cells or tissue, a piece of leaf or other plant part wasremoved from a modified and control (non-modified) Brassica plant. Theleaf or plant part was then examined with a fluorescencestereo-microscope using 20-100× magnification and a rhodamine filterset.

EXAMPLE 7 Tomato Centromere Discovery and Mini-Chromosome Construction

BAC Library Construction

A Bacterial Artificial Chromosome (BAC) library was constructed fromTomato genomic DNA isolated from Tomato variety “Microtom” and digestedwith the restriction enzyme MboI. This enzyme was chosen because it ismethylation insensitive and therefore can be used to enrich BAClibraries for centromere DNA sequences.

Probe Identification and Selection

Tomato repetitive genomic or plastid sequences, including specificcentromere-localized sequences, were initially compiled as candidateprobes for hybridization with the BAC libraries. These probesrepresented various classes of Tomato repetitive sequences includingsatellite repeats (heterochromatic/centromere-specific), rDNA, andhypermethylated and highly repetitive DNA fractions.

Six probes were picked to interrogate the BAC libraries. These probesrepresent different groups of commonly found repetitive sequences in theTomato genome. The probes selected are shown in Table 24 and were LESAT(the Tomato centromere satellite, in two different variants; SEQ ID NOS:37 and 38), a tomato microsatellite (LEGATAREP; SEQ ID NO: 39), HpaII(bulk methylated DNA purified from genomic DNA by failure to digest withthe methylation-sensitive enzyme HpaII), bulk repetitive DNA purifiedfrom genomic DNA by reassociation kinetics (Cot), and telomere. Theprobes were prepared by PCR, from cloned fragments, or from bulkmethylated or repetitive DNA prepared from Tomato genomic DNA. Thetelomere probe sequence (SEQ ID NO: 40) was generated by PCR using thefollowing primers: CHHZ-97 (AGGCGCGCCACCTGCAGGA GAGCTCGGTCTCA TCGAGACAC;SEQ ID NO: 41) and CHHZ-98 (GGTCGACGGCCCGGGCGTT TAAACCCGGGCTCAC; SEQ IDNO: 42). Probes were prepared and labeled with standard molecularbiology methods. TABLE 24 Tomato Repetitive Sequences and BAC LibraryProbes group clone used for GenBank group # name probe name Descriptionhyb accession* 1 centromere TC2 LESAT, tomato 5012-5-11-C02 X87233.1repeat (SEQ ID NO: 37) centromere satellite (different variant) TE1LESAT, tomato 5012-5-11-E01 X87233.1 (SEQ ID NO: 38) centromeresatellite (different variant) 2 microsatellite TE12 LEGATAREP,5012-5-11-E12 X90937.1 repeat (SEQ ID NO: 39) tomato microsatelliterepeat 3 bulk TCot6 Purified repetitive N/A N/A repetitive DNA fractionDNA T HpaII Purified N/A N/A methylated DNA fraction 4 telomere TTelTelomere PCR product N/A (SEQ ID NO: 40)*Accession number of BLAST hit; actual sequence has not been depositedin GenbankLibrary Interrogation and Data Analysis

The BAC clones from the libraries were spotted onto filters and thesefilters were hybridized with each of the probes to identify specific BACclones that contain DNA from the group of sequences represented by theprobe(s).

A total of 18,432 BAC clones from the library were interrogated witheach of the probes described above sing the following hybridizationconditions: 0.5×SSC 0.25% SDS at 65 degrees for 15 minutes, followed bya wash at 65 degrees for a half hour. The hybridization intensities ofthe BAC clones with each probe were scanned to quantitate hybridizationintensity for each clone. The outputs (scores of 1 to 10 based on thehybridization intensities, with 10 being the highest intensity) wereimported into a relational database, for further analysis andclassification. The database contained a total of five tables that wereused for BAC selection. Each table contains a total of 18,432 entries:the hybridization scores of each BAC clone from the library to one ofthe probes used to interrogate the library. Data analysis was done usingstandard SQL (Structured Query Language) routines to find BACs thatcontain different groups of repetitive sequences.

Classification and Selection of BAC clones from Mini-ChromosomeConstruction

BAC clones containing centromeric/heterochromatic DNA were identified bytheir hybridization scores to different probes. The goal was to selectBAC clones that contained a diverse set of various repetitive sequences.Eleven classes of centromeric BAC clones, some of which overlap, wereeventually chosen to cover the broadest possible range ofcentromeric/heterochromatic sequences for mini-chromosome construction.Detailed descriptions of each class and probe hybridization values foreach class are shown in Table 25. TABLE 25 Classification of tomato BACclones containing centromeric DNA Probe Hybridization Range Class LESATLEGATA Hpa II # clones Class Properties LESAT C2 E1 REP E12 (METH) TELidentified A High N/A >=6 >=6 >=6 N/A 30 Meth, E1, E12 B High N/A >=7N/A >=7 >=7 36 Meth, E1, TEL C High <=5 N/A N/A >=7 >=7 9 Meth, TEL; lowC2 D High N/A >=8 N/A >=7 N/A 103 Meth and E1 E High N/A N/A >=6 >=6 N/A35 Meth and E12 F High E1 N/A >=6 >=6 N/A N/A 75 and E12 G High E1N/A >=8 N/A N/A >=8 8 and TEL H High E1 N/A >=8 <=4 <=6 N/A 89 only IHigh TEL N/A <=4 N/A <=4 >=8 49 only J High N/A N/A <=4 >=7 <=4 15 Methonly K High E12 N/A N/A >=7 <=4 <=4 2 only Total** 451*Values represent hybridization intensities of an individual BAC to eachprobe on a scale of 1 to 10. Values were normalizedN/A = not applicable

A number of representative clones from each class were chosen to yield atotal of 278 BAC clones for further analysis by restriction digestfingerprinting. The BAC clones were fingerprinted (Table 26) based onrestriction sites found in the centromere specific sequence(s) asdescribed in Example 1. The restriction enzyme HinfI was used to digestthe BAC clones. After fingerprinting, 100 BACs were selected for furthertesting using the method described in Example 1.

L. esculentum (tomato) BAC TB99 was deposited with the American TypeCulture Collection (ATCC) P.O. Box 1549 Manassas, Va. 20108, USA on Feb.23, 2005 and assigned Accession No.

Thirty BAC clones (from the original 278) were selected formini-chromosome construction based on the fingerprint class which wasdefined as a simple or complex laddering pattern. Table 26 lists thefingerprint patterns for a selected set of 26 Tomato BAC clones. Tomatofingerprints were classified into 3 classes: 1. high complexity(multiple large bands with no indication of laddering), 2. low ladder(predominant bands at multiples of the unit repeat size for thecentromere satellite, and 3. complex ladder (features of both previoustypes). Table 27 lists the fingerprint classes for 7 selected tomatoBACs. The preferred BACS have an Table 27 lists the fingerprint classesfor 11 selected Brassica BACs. TABLE 26 Restriction EndonucleaseFingerprinting of 26 Tomato BACs BAC BAC Fingerprint Number Class ClassProperties Class* MiniC tested TB1 G Hi LE SAT/Tel 2. Low ladder TB1R4-3TB4* G Hi LE SAT/Tel 2. Low ladder TB4R1-2 TB6 J Hi Hpa only 1. complexTB6R4-1 TB10 I Hi Tel only 1. complex TB10R4-1 TB12 I Hi Tel only 1.complex TB12R1-1 TB16 F Hi LESAT/LE Gata rep 2. Low ladder TB16R4-5 TB17J Hi Hpa only 1. complex TB17R1-1 TB21 D Hi Hpa/LESAT 2. Low ladderTB21R1-2 TB22 D Hi Hpa/LESAT 2. Low ladder TB22R1-1 TB23* G Hi LESAT/Tel2. Low ladder TB23R1-5 TB29 J Hi Hpa only 3. TB29R1-1 complex/ladderTB47 D Hi Hpa/LESAT 2. Low ladder TB47R1-1 TB55 B Hi LESAT/Hpa/TEL 2.Low ladder TB55R1-5 TB56 D Hi Hpa/LESAT 3. TB56R1-3 complex/ladder TB67H Hi LESAT only 2. Low ladder TB67R1-1 TB72 H Hi LESAT only 3. TB72R1-3complex/ladder TB73 D Hi Hpa/LESAT 2. Low ladder TB73R1-2 TB80* D HiHpa/LESAT 3. TB80R1-2 complex/ladder TB82* H Hi LESAT only 2. Low ladderTB82R1-4 TB91 H Hi LESAT only 2. Low ladder TB91R1-2 TB92 H Hi LESATonly 3. TB92R1-3 complex/ladder TB99* H Hi LESAT only 2. Low ladderTB99R1-5 TB101* B Hi LESAT/Hpa/TEL 2. Low ladder TB101R1-5 TB114 H HiLESAT only 2. Low ladder TB114R1-1 TB115 H Hi LESAT only 2. Low ladderTB115R4-2 TB132* F Hi LESAT/LE Gata rep 2. Low ladder TB132R1-3

TABLE 27 Restriction endonuclease fingerprint classification for 7selected tomato BACs BAC Hyb Number Class Class Properties FingerprintClass TB4 G Hi LE SAT/Tel 2. Low ladder TB23 G Hi LESAT/Tel 2. Lowladder TB80 D Hi Hpa/LESAT 3. complex/ladder TB82 H Hi LESAT only 2. Lowladder TB99 H Hi LESAT only 2. Low ladder TB101 B Hi LESAT/Hpa/TEL 2.Low ladder TB132 F Hi LESAT/LE Gata 2. Low ladder repConstruction of Mini-Chromosomes

For each BAC identified above, a mini-chromosome was constructed using aCre-Lox Recombination-Donor vectors as described in Example 2. Tomatomini-chromosomes were constructed from a total of 30 BACs using donorvector 151 and 153 in this assembly process, and were subsequentlytested in several different tomato cell lines. Mini-chromosome geneticelements within the pCHR151 and pCHR153 vector are set out in Tables 10and 28. TABLE 28 Donor Components of pCHR153 Size Genetic Element (basepair) Location (bp) Details EF1α A3 Promoter 2051  361-2411 Arabidopsisthaliana elongation factor 1 alpha A3 promoter (At1g07940) DsRed2 + NLS780 2448-3227 Nuclear localized red fluorescent protein from Discosomasp. (Matz, M et. al Nat Biotechnol 1999 Dec; 17(12): 1227). Pyruvatekinase 332 3362-3693 Arabidopsis thaliana pyruvate terminator kinaseterminator (At5g52920) Bacterial Kanamycin 817 3838-4654 Bacterialkanamycin selectable marker Act2 terminator 800 4836-5635 Arabidopsisthaliana Actin 2 terminator MerA 1695 5789-7483 Plant selectable markerproviding resistance to mercuric ions (Rugh et. al. PNAS 1996 93: 3182)Act2 promoter + intron 1482 7486-8967 The Arabidopsis thaliana promoterActin 2 plus natural intron LoxP 34 312-345 & 8984-9017 Recombinationsite for Cre mediated recombination (Arenski et. al 1983, Abremski et.al 1984)

EXAMPLE 8 Testing of Tomato Mini-Chromosomes and Regeneration of TomatoPlants Modified with Mini-Chromosome DNA

The biolistic delivery method using wet gold particles, described inExample 2, was used to deliver mini-chromosomes to tomato cells.Functional testing of mini-chromosomes using transient assays asdescribed in Example 3. In the transient assay, mini-chromosomes weredelivered to tomato cells of a population that is undergoing celldivision, in this case tomato suspension cells grown in liquid cultureor callus cells grown on plates. PC703, a publicly available tomatocallus cell line, was routinely used in transient assays describedabove. However, any actively dividing cell type can be used for thisassay, such as root tissue, meristem tissue, or callus derived from anyplant tissue.

After DNA delivery, the cell population was then monitored forfluorescent protein expression over the course of one to several weeks.Mini-chromosomes containing active centromeres allowed the formation offluorescent cell clusters, which are derived from a single progenitorcell that has divided and passed the mini-c to its daughter cells.Accordingly, single fluorescent cells and clusters of fluorescent cellsof various sizes were scored in the growing cell population after DNAdelivery. A number of stable cell lines were obtained following thedelivery of the mini-chromosomes listed in Table 29. TABLE 29 PreferredChromatin tomato BACs, centromeres (CEN), and mini- chromosomes (MC)based upon transient expression assays and generation of stable tomatocell lines. Stable BAC CEN MC clones Number Number Number BAC Classgenerated TB4 TB4 TB4R1-2 Hi LE SAT/Tel yes TB23 TB23 TB23R1-5 HiLESAT/Tel yes TB80 TB80 TB80R1-2 Hi Hpa/LESAT yes TB82 TB82 TB82R1-4 HiLESAT only yes TB99 TB99 TB99R1-5 Hi LESAT only yes TB101 TB101TB101R1-5 Hi yes LESAT/Hpa/TEL TB132 TB132 TB132R1-3 Hi LESAT/LE yesGata rep

To obtain trans-chromosomal tomato plants, the promising centromeresidentified above were combined with a different set of genes than thosepresent in donor vector 151 or 153 which were used in the constructionof the initial set of 26 mini-chromosomes. The mini-chromosomeconstruction procedure was thus repeated for BACs TB99 and TB132 usingdonor vectors 487-489 (See Example 2 and Tables 12-14 for description)and 531 (see Table 30 below for descriptions of the 531 donor vector),following the same steps as described above. Five mini-chromosomes wereobtained that contain nptII, summarized below: TABLE 30 Donor Componentsof pCHR531 Size Genetic Element (base pair) Location (bp) Details UBQ10promoter 359  361-2398 Arabidopsis thaliana polyubiquitin promoter(At4g05320) DsRed2 + NLS 780 2435-3214 Nuclear localized red fluorescentprotein from Discosoma sp. (Matz, M et. al Nat Biotechnol 1999 Dec;17(12): 1227). Pyruvate kinase 332 3349-3680 Arabidopsis thalianapyruvate terminator kinase terminator (At5g52920) Bacterial Kanamycin817 3825-4641 Bacterial kanamycin selectable marker Act2 terminator 8004823-5622 Arabidopsis thaliana Actin 2 terminator NptII 795 5685-6479Neomycin phosphotransferase II plant selectable marker UBQ10 intron 3596507-6865 PCR amplified Arabidopsis thaliana intron from UBQ10 gene(At4g05320) for stabilization of NptII gene transcript and increaseprotein expression levels Pgd Fly promoter 2140 6873-9012 PCR amplifiedpromoter of phosphogluconate dehydrogenase gene from Drosophilamelanogaster LoxP 312-345 & 9029-9062 Recombination site for Cremediated recombination (Arenski et. al 1983, Abremski et. al 1984)

Promoter driving Mini-C Cen name Cen size Donor vector nptII* TB99R7-1TB99 50 kb pCHR487 Tef2 TB99R8-1 TB99 15 kb pCHR488 GPD-1 TB99R10-1 TB9948 kb pCHR531 Pgd-1 TB132R8-1 TB132 48 kb pCHR488 GPD-1 TB132R10-2 TB13227 kb pCHR531 Pgd-1

For tomato modification with these mini-chromosomes (derived from TB99and TB132 combined with donor vectors 487, 488 and 531), the followingprocedure was developed. Tomato seeds were surface sterilized in 10%bleach for 15 minutes and washed 4 times with sterile distilled water.The seeds were placed in sterile Petri dishes and dried under sterileair flow in a tissue culture hood. The seeds were germinated in magentacontainers on solid medium (0.5× MS salts, 1× MS vitamins, 10 g/lsucrose, 8 g/l tissue culture agar, 0.5 mM MES, 1.3 g/liter calciumgluconate, pH 5.8) for 8 days at ambient temperature under lights.

Cotyledons and hypocotyls were removed from the seedlings for explants.The cotyledon pieces were cut into slices approximately 3-4 mm wide andthe hypocotyls were cut longitudinally. Both types of explants weregrown on preculture medium (1× MS salts, 1× MS vitamins, 3% sucrose, 1mM MES, 8 g/liter tissue culture agar; pH 5.7-5.8) The medium alsocontained either 1 mg/l BA+0.1 mg/l non-essential amino acids or 0.75mg/l zeatin+1 mg/l IAA. The cotyledon pieces were cultured with theabaxial side in contact with the medium; while the hypocotyls pieceswere cultured with the wounded side away from the medium. The explantswere pre-cultured for 3-6 days under lights.

The explants were then transferred to 5 cm polyethylene mesh circles andbombarded using the wet biolistics method as described in Example 2. Thesame surface was bombarded as facing upwards (away from the medium)during pre-culturing. After bombardment, the explants were transferredback onto preculture medium and kept under light.

Two days after bombardment, the explants were transferred ontopreculture medium containing 100 mg/liter kanamycin. The explants werecultured on this medium under light for 6 weeks to three months withsub-culturing onto fresh medium every 3 weeks. Starting at 6 weeks afterthe onset of selection, the explants were screened with a fluorescencestereomicroscope for appearance of fluorescent calli or shoots.

The presence of fluorescent protein expression was detected as describedin Example 3. Fluorescent calli or shoots were removed from the explantsand transferred to plates with MS3 basal medium (1× MS salts, 1×Gamborg's vitamins, 3% sucrose, 0.5 mM MES, 8 g/liter tissue cultureagar, pH 5.8)+0.75 mg/l zeatin. The calli were grown on this mediumuntil visible shoots formed. The shoots arising directly from thekanamycin plates were kept on this medium for only 1-2 weeks. The shootswere then transferred to MS3 basal medium+0.1 mg/liter zeatin and weresubcultured on this medium until the shoots elongated (1-3 cm shootlength, at least 5 mm of stem length), with medium changes every 2weeks.

The elongated shoots were transferred to magenta containers containing0.5× MS salts, 1× MS vitamins, 1% sucrose, 0.1 mg/liter IBA, 1 mM MES,pH 5.7-5.8. Rooting was allowed to proceed until well-formed rootsgenerated (2 weeks to 2 months). Plantlets were then transferred tosoil. TABLE 31 lists the number of trans-chromosomal events for tomatoand tobacco. Construct # events Table 31a Tobacco transformants withtomato mini-C's - summary of events TB99R7-1  9 TB99R10-1  3 TB132R10-2 2 TB99R8-1  1 TB132R8-1  2 Table 31b Tomato transformants with tomatomini-C's - summary of events TB99R7-1  13* TB132R10-2  10**not all events fully regenerated, some of them still in organogenicphase

EXAMPLE 9 Regeneration of Tobacco Plants Modified with Mini-ChromosomeDNA

The biolistic delivery method using wet gold particles, described inExample 2, was used to deliver tomato mini-chromosomes (described inExample 7) to Tobacco cells.

An explant from a tobacco leaf was cut using a cork borer. The leaveswere immersed in MS medium during cutting to avoid tissue dehydration.The leaf disks were placed adaxial side up onto plates containing callusinducing medium with vitamins (4.44 g/L MS Basal Medium w/GamborgVitamins, 0.5 g/L MES, 3% sucrose, 0.5% tissue culture agar,non-essential amino acids, kinetin and 4 ml of 1000× Gamborg Vitamins,pH 5.8). After four days, the explants were bombarded with wet goldparticles/DNA suspension as described in Example 2 using 450 psi rupturedisks with the sample tray in the lowest position. For bombardment,explants were transferred onto 50 mm polyethylene mesh circles, andcovered with mosquito netting.

Immediately after bombardment, all explants were returned to theiroriginal plates for 24 hours. Subsequently, the explants weretransferred to MBNV plates (4.44 g/L MS Basal Medium w/Gamborg Vitamins,0.5 g/L MES, 3% sucrose, 0.5% tissue culture agar, 0.1 mg/L NAA, 2.0mg/L BA, 4 ml of 1000× Gamborg Vitamins pH 5.8) containing 50 μg/ml ofkanamycin. After 5 days of selection, the explants were transferred tofresh MBNV plates containing 100 μg/ml kanamycin for 10 days.Subsequently, the explants were transferred to MBN plates (MBNV platesdescribed above but without the added vitamins; 1× final concentrationof Gamborg's vitamins), containing 100 μg/ml of kanamycin. These plateswere subsequently subcultured about every 2 weeks afterwards, onto thesame MBN plates containing 100 μg/ml of kanamycin.

The presence of fluorescent protein expression was detected as describedin Example 3. A pea-sized fluorescent calli was removed from the plateand transferred to MBN medium without kanamycin. Fluorescent shoots wereremoved from the callus as they developed, and these shoots weretransferred to Magenta containers containing 1× MS salts, 1× MS vitaminsand 2% sucrose, pH 5.8. As the shoots enlarged and root formed, theywere transferred to Magenta containers containing 0.5× MS salts, 0.5× MSvitamins and 1% sucrose. The transchromosomal events for tobacco plantsare described above in Table 31.

EXAMPLE 10 Soybean Centromere Discovery and Mini-Chromosome Assembly andConstruction

BAC Library Construction

A Bacterial Artificial Chromosome (BAC) library was constructed fromSoybean genomic DNA isolated from Glycine max variety “Williams 82” anddigested with the restriction enzyme MboI. This enzyme was chosenbecause it is methylation insensitive and therefore can be used toenrich BAC libraries for centromere DNA sequences.

Probe Identification and Selection

Five groups of soybean repetitive genomic or plastid sequences,including specific centromere-localized sequences, were initiallycompiled as candidate probes for hybridization with the BAC libraries(Table 32). These probes represented various classes of Soybeanrepetitive sequences including satellite repeats(heterochromatic/centromere-specific), retroelements, telomeres, rDNA,and hypermethylated DNA fractions.

Seven probes were picked to interrogate the BAC libraries. These probesrepresent different groups of commonly found repetitive sequences in theSoybean genome. The probes selected are shown in Table 32 and were: twovariants of the soybean centromere satellite (TRS and 3X1), 5S ribosomalDNA, plant telomeres, HpaII (bulk methylated DNA purified from genomicDNA by failure to digest with the methylation-sensitive enzyme HpaII)and Sau3A (bulk methylated DNA purified from genomic DNA by failure todigest with the methylation-sensitive enzyme Sau3A), and retroelement.The probes were prepared from cloned fragments isolated or from bulkmethylated DNA prepared from Soybean genomic DNA. Sequences from the orPCR primes clones used to prepare each probe are shown in Table 32. Thetelomere probe sequence (SEQ ID NO: 40) was generated by PCR using thefollowing primers: CHHZ-97 (AGGCGCGCCACCTGCAGGAGAGCTCGGTCTCA TCGAGACAC;SEQ ID NO: 41) and CHHZ-98 (GGTCGACGGCCCGGGCGTT TAAACCCGGGCTCAC; SEQ IDNO: 42). Probes were prepared and labeled with standard molecularbiology methods. TABLE 32 Soybean Genetic Repetitive Sequences and BACLibrary Probes Group Group # Name Probe Name Description Clone used forhyb GenBank accession* 1 Cen repeat SC2 TRS (centromere satellite5012-5-9-C02 gi|11464861|gb|AF297984.1|AF297984 (SEQ ID NO: 43) repeatvariant) Glycine max clone TRS2 gi|11464862|gb|AF297985.1|AF297985Glycine max clone TRS3 gi|11464860|gb|AF297983.1|AF297983 Glycine maxclone TRS1 (SEQ ID NO: X) SE7 3X1(centromere satellite 5012-5-9-E07Z26334.1|GMP3X1SAT (SEQ ID NO: 44) repeat variant) (SEQ ID NO: X) 2 rDNASC11 5S rDNA 5012-5-9-C11T X06044.1|GMRN45SI Soybean (SEQ ID NO: 45)4.5 - 5S rRNA intergenic (SEQ ID NO: X) 3 retroelement SG12retrovirus-like element 5012-5-9-G12T AF186186 (SEQ ID NO: 46) (SEQ IDNO: X) 4 bulk SHpaII Purified methylated DNA N/A N/A repetitive fractionDNA SSau Purified methylated DNA N/A N/A fraction 5 telomere StelTelomere PCR product N/A (SEQ ID NO: 40)*Accession number of BLAST hit; actual sequence has not been depositedin GenbankLibrary Interrogation and Data Analysis

The BAC clones from the libraries were spotted onto filters and thesefilters were hybridized with each of the probes to identify specific BACclones that contain DNA from the group of sequences represented by theprobe(s).

A total of 18,432 BAC clones from the library were interrogated witheach of the probes described above sing the following hybridizationconditions: 0.5×SSC 0.25% SDS at 65 degrees for 15 minutes, followed bya wash at 65 degrees for a half hour. The hybridization intensities ofthe BAC clones with each probe were scanned to quantitate hybridizationintensity for each clone. The outputs (scores of 1 to 10 based on thehybridization intensities, with 10 being the strongest intensity) wereimported into a relational database, for further analysis andclassification. The database contained a total of seven tables. Eachtable contains at total of 18,432 entries: the hybridization scores ofeach BAC clone from the library to one of the probes used to interrogatethe library. Data analysis was done using standard SQL (Structured QueryLanguage) routines to find BACs that contain different groups ofrepetitive sequences.

Classification and Selection of BAC Clones for Mini-ChromosomeConstruction

BAC clones containing centromeric/heterochromatic DNA were identified bytheir hybridization scores to different probes. The goal was to selectBAC clones that contained a diverse set of various repetitive sequences.Twelve classes of centromeric BAC clones, some of which overlap, wereeventually chosen to cover the broadest possible range ofcentromeric/heterochromatic sequences for mini-chromosome construction.Detailed descriptions of each class and probe hybridization values foreach class are shown in Table 33. TABLE 33 Classification of Soybean BACclones containing centromeric DNA Probe Hybridization Range 5S Class TRS3X1 rDNA RE Meth Meth # clones Class Properties (C2) (E7) (C11) (G12)(HpaII) (Sau3A) TEL identified A High 3X1 <=4 >=10  N/A N/A >=1 N/A N/A155 B High TRS >=10  <=4 N/A N/A >=1 N/A N/A 114 C High <=10  <=10  N/AN/A >=7 N/A N/A 43 HpaII D High <=4 <=4 N/A N/A >=5 N/A N/A 44 HpaIIonly E High TRS >=6 N/A N/A N/A >=5 N/A N/A 34 and HpaII F HighestN/A >=6 N/A N/A >=5 N/A N/A 103 3X1 and High HpaII G High 3X1 N/A >=6N/A N/A >=5 N/A N/A 103 and Highest HpaII H High TRS >=8 >=8 N/A N/A >=1N/A N/A 54 and 3X1 I High >=7 >=7 N/A N/A >=4 N/A N/A 5 TRS, 3X1, HpaIIJ High >=6 >=6 N/A N/A >=4 N/A N/A 33 TRS, 3X1, HpaII K High TEL >=1 >=1N/A N/A >=1 N/A >=8 6 L High RE >=1 >=1 N/A >=8 >=1 N/A N/A 105 Total**642N/A = not applicable; This is functionally equivalent to >=1, as well as<=10 Classes F and G have the same threshold values but the selectedclones for class F show the highest 3X1 scores of all the clones in theclass; For class G, the selected clones show the highest HpaII scoresfor the class., and Classes I and J have the same criteria, but slightlydifferent thresholds.

A number of representative clones from each class were chosen to yield atotal of 230 BAC clones for further analysis by restriction digestfingerprinting. The BAC clones were fingerprinted (Table 34) based onrestriction sites found in the centromere specific sequence(s) asdescribed in Example 1. The restriction enzymes HinfI and DdeI were usedto digest the BAC clones. After fingerprinting, 33 BACs were selectedfor further testing using the method described in Example 1.

Thirty-three BAC clones (from the original 230) were selected formini-chromosome construction and testing based on the fingerprint classwhich was defined as a simple or complex laddering pattern. Soybeanfingerprints were classified into 3 classes: 1. high complexity(multiple large bands with no indication of laddering), 2. low ladder(predominant bands at multiples of the unit repeat size for thecentromere satellite, and 3. complex ladder (features of both previoustypes).Table 34 lists the fingerprint patterns for this selected set ofSoybean mini-chromosomes. The preferred BACs have an *. Table 35 liststhe fingerprint classes for 10 selected soybean BACs. TABLE 34Restriction endonuclease fingerprinting of 33 soybean BACs HinfI DdeIBAC BAC Class Fingerprint Fingerprint Number Class Properties ClassClass MiniC tested SB1 J High TRS, n/d* 4. 6 bands/9 SB1R3-1 3X1, HpaIIbands SB2 D High HpaII 4. 6 bands/ 4. 6 bands/9 SB2R5-1 only 9bandsbands SB3* H High TRS 3. complex 3. complex SB3R1-1 and 3X1 ladderladder SB6* B High TRS 2. simple 2. simple SB6R15-3 ladder ladder SB8 AHigh 3X1 1. complex 1. complex SB8R3-1 SB9* H High TRS 3. complex 2.simple SB9R8-1 and 3X1 ladder ladder SB10 L High RE 1. complex 1.complex SB10R4-1 SB11* B High TRS 3. complex 3. complex SB11R3-1 ladderladder SB11R3-2 SB11R3-3 SB12* B High TRS 3. complex n/d* SB12R2-1ladder SB12R2-2 SB12R2-3 SB21 K High TEL 2. simple 1. complex SB21R1-2ladder SB22* A/L High 2. simple 1. complex SB22R2-1 3X1/RE ladder SB24A/L High 2. simple 1. complex SB24R2-3 3X1/RE ladder SB29 B High TRSn/d* 3. complex SB29R2-2 ladder SB38* H High TRS n/d* 3. complexSB38R2-1 and 3X1 ladder SB38R2-2 SB41 H High TRS n/d* 2. simple SB41R3-1and 3X1 ladder SB45 J High TRS, 4. 6 bands/ 4. 6 bands/ SB45R5-1 3X1,HpaII 9bands 9bands SB50* B High TRS 3. Complex n/d* SB50R1-1 ladderSB62 A High 3X1 2. Simple n/d* SB62R1-2 ladder SB93 B High TRS 3.complex n/d* SB93R3-2 ladder SB93R3-3 SB97 A High 3X1 2. simple n/d*SB97R3-2 ladder SB102 A High 3X1 2. simple n/d* SB102R3-1 ladder SB107 BHigh TRS 2. simple n/d* SB107R3-1 ladder SB111 A High 3X1 2. simple n/d*SB111R3-1 ladder SB112 A High 3X1 3. complex n/d* SB112R3-1 ladderSB116* A High 3X1 2. simple n/d* SB116R3-1 ladder SB118 A High 3X1 2.simple n/d* SB118R3-1 ladder SB119 A High 3X1 2. simple n/d* SB119R3-2ladder SB123 A High 3X1 2. simple n/d* SB123R3-2 ladder SB125* B HighTRS 3. complex n/d* SB125R3-1 ladder SB135 B High TRS 2. simple n/d*SB135R3-2 ladder SB138 A High 3X1 2. simple n/d* SB138R3-1 ladder SB178B High TRS 2. simple n/d* SB178R3-1 ladder SB219 B High TRS 2. simplen/d* SB219R3-3 ladder

TABLE 35 Restriction endonuclease fingerprint classification for 10selected soybean BACs BAC Class Fingerprint Class Number ClassProperties HinfI DdeI SB3 H High TRS and 3. complex 3. complex 3X1ladder ladder SB6 B High TRS 2. simple ladder 2. simple ladder SB9 HHigh TRS and 3. complex 2. simple ladder 3X1 ladder SB11 B High TRS 3.complex 3. complex ladder ladder SB12 B High TRS 3. complex n/d* ladderSB22 A/L High 3X1/RE 2. simple ladder 1. complex SB38 H High TRS andn/d* 3. complex 3X1 ladder SB50 B High TRS 3. Complex n/d* ladder SB116A High 3X1 2. simple ladder n/d* SB125 B High TRS 3. complex n/d* ladder

G. Max (soybean) BAC SB6 was deposited with the American Type CultureCollection (ATCC) on P.O. Box 1549 Manassas, Va. 20108, USA on February23, 2005 and was assigned Accession No. ______.

Construction of Mini-Chromosome

Each of the soybean BAC clones identified above were constructed using aCre-Lox Recombination-Donor as described in Example 2. Soybeanmini-chromosomes were constructed from a total of 33 BACs using donorvector pCHR151 in this assembly process, and were subsequently tested inseveral different soybean cell lines. Mini-chromosome genetic elementswithin the pCHR151 are described above in Table 10. The Soybeanmini-chromosomes were used to transform broccoli plants (see Table 37below).

Identification of Functional Soybean Centromeres

Functional testing of mini-chromosomes using transient assays asdescribed may be carried out as in Example 3. Mini-chromosomes aredelivered to the soybean cells using wet biolistic as described inExample 2. After DNA delivery, the cell population is then monitored forfluorescent protein expression over the course of one to several weeks.Mini-chromosomes containing active centromeres will allow the formationof fluorescent cell clusters, which are derived from a single progenitorcell that has divided and inherited the mini-chromosome to its daughtercells. Accordingly, single fluorescent cells and clusters of fluorescentcells of various sizes are scored in the growing cell population afterDNA delivery. Standard protocols for soybean tissue culture andtransformation, including those available at the University of Iowa,School of Agriculture web site, are used to regenerate adchromosomalsoybean plants.

Mini-Chromosome Autonomy.

As a direct demonstration of mini-chromosome autonomy, circularconstructs were recovered from fluorescent soybean cell lines that hadbeen propagated for 5 months (˜25 generations) following bombardment.Genomic DNA was extracted from a cell line containing SB12MC, and theDNA was treated with a highly processive exonuclease, resulting indegradation of all linear DNA fragments including those derived fromhost chromosomes. Surviving DNA molecules were introduced into E. coliand transformants were selected on antibiotic-containing medium.

Genomic DNA from unmodified soybean cells did not result in anyantibiotic-resistant colonies, while DNA purified from the linecontaining mini-chromosomes yielded 13 independent modified colonies (2from exonuclease-treated DNA and 11 from untreated DNA, R1-R13). DNA wasextracted from each transformed E. coli clone and characterized by gelelectrophoresis and sequencing. While the vector backbone of the rescuedmini-chromosomes was typically unchanged (9/13 transformants). BAC-endsequencing demonstrated that 11/13 of the recovered clones retained thesame DNA sequence junctions at the centromere cloning boundaries as theparental molecule (600/600 bp sequenced at each junction), including twoof the mini-chromosomes with altered vector sequences.

Fluorescence in situ hybridization (FISH) was carried out to examinemini-chromosome autonomy and copy number. Cells containingmini-chromosomes were arrested in metaphase, spread on slides and probedwith labeled soybean centromere satellite DNA (red) and mini-chromosomevector sequences. In cells hybridizing to both the centromere and vectorprobes, only one autonomous mini-chromosome was identified; similarsignals were not detected in non-transgenic controls. Only a subset ofthe native centromeres were labeled, suggesting that the satellitesequence used as a probe is chromosome-specific. Strong vectorhybridization signals were not detected within the host chromosomes,providing further evidence that the mini-chromosome DNA remainedautonomous.

Satellite Sequences from Mini-Chromosomes

The identified soybean mini-chromosomes defined DNA sequences sufficientfor centromere activity. The sequence content of thecentromere-containing BAC clones and the mini-chromosomes derived fromthem with quantitative dot blots, using probes that correspond to i)vector sequences, ii) soybean satellites, iii) the SIRE retroelement,and iv) 28S rDNA, all of which are highly repetitive sequences presentin the soybean centromeric region. BAC SB1 lacked centromere activityand has a high rDNA content with undetectable satellite and retroelementsequences. By contrast, the mini-chromosome derivatives of SB6 and SB12had similar compositions, with 6.4 and 11.8 kb of centromere satellite,respectively. The recovered SB12 derivatives retained the parentalcomposition (R4, R6, R7, R10), had a two-fold decrease in satellite (R1,R2, R3, R5), or had little or no satellite (R8, R9, R11, R12, R13). TheSIRE retroelements present in SB12 were retained in most of thederivatives, suggesting little selective pressure to eliminate thissequence during growth of the modified cell culture. In addition, eachmini-chromosome also contained ˜8.5 kb of gene sequence from pCHR151(Table 10).

DNA sequencing of SB12R2-3 (1.4-fold insert coverage) revealed ˜80% ofthe insert was composed of tandem satellite repeats (Genbank U11026 andZ26334), ˜9.9% was made up of retroelement-related sequences, and ˜10.1%represented novel, contiguous sequence. The same analysis also produced1.6-fold vector sequence coverage, indicating little if any cloning biasagainst fragments from the centromere. Individual satellite repeatsshowed an average of 91.3% (s.d.=11.3%) identity to each other, withspecific regions showing significantly higher and lower levels ofvariability. Comparing the satellite repeat consensus from SB12R2-3 tothat obtained from random satellite sequences (CrGm1 and CrGm2)identified several bases that differed significantly (χ² test, P <0.05).The SB12R2-3 satellite repeats showed an average length of 91.07±0.40bp, similar to the CrGm2 91-base consensus and differing from the CrGm192-base consensus. FIG. 6 shows an alignment of these consensussequences.

EXAMPLE 11 Analysis of Mini-Chromosomal DNA Expression in TransgenicPlants

Visual Scoring

The adchromosomal plants described above in Example 4 (broccoli),Example 5 (canola), Example 6 (tobacco) and Example 7 (tomato) weretested to determine if the mini-chromosome DNA was being expressed. Thepresence of visible marker genes allowed for visual analysis todetermine if the regenerated plants were expressing the fluorescentprotein present on the mini-chromosome. For visual analysis, a piece ofa leaf or other plant part was cut from the adchromosomal plant. Asimilar part is cut from a control plant (non-adchromosomal). The plantswere analyzed under a fluorescence stereo-microscope as described inExample 3.

Table 37 displays the results of visual scoring of T0 adchromosomalplants. TABLE 37 No. of Centromeres Tested Host and Origin of CentromereNo. of Positives Scored In Broccoli 15 (13 broccoli, 1 soybean 12 (10broccoli, 1 plants and 1 tobacco) soybean and 1 tobacco) Broccoli 15 (13broccoli, 1 soybean 14 (12 broccoli, 1 cluster and 1 tobacco) soybeanand formation 1 tobacco) Canola  1 (broccoli)  1 plant Canola 34(broccoli) 10 cell culture Tobacco  2 (tomato)  2 plants Tomato  8(tomato)  2 plants Tomato 20 (tomato)  2 cell culture

Expression of the fluorescent protein gene encoded by themini-chromosome was readily observed in the cells of a piece of tissuesuch as leaf or root taken from a transchromosomal plant under afluorescence stereo-microscope. Fluorescence was very high and uniformthroughout the tissue. Sectoring of fluorescence protein expression wasobserved in some cases.

PCR Analysis

PCR was used to amplify sequences within the mini-chromosome. Thismethod allowed for detection of all mini-chromosome parts or a subset ofparts. PCR analysis was also carried out in DNA samples isolated fromwhole cell DNA preparations from adchromosomal broccoli, canola andtobacco plants. A piece of leaf was cut from the plant and ground byhand using a pestle and a microcentrifuge tube. The DNA was isolatedusing Qiagen Kit (catalog no. 69106) according to the manufacturer'sinstructions. PCR reactions were carried out as follows: 3 μl templateDNA, 2.5 μl of 10× Termopol buffer (New England Bioscience, Catalog No.B9004S), 0.5 μl dNTP's (10 mM each), 1.5 μl oligo 1 (20 μM), 1.5 μloligo 2 (20 μM), 15.5 μl dH₂O and 0.5 μl Taq polymerase (New EnglandBioscience, Catalog No. M0267S). The Oligos used either detected theDsRed gene (CHHZ 150 and 152) or the UBQ10 promoter (CHHZ 467 and 469).To detect DsRed oligo CHHZ 150 (TGAACGGCCACGAGTTCGAGATCG; SEQ ID NO: 47)and oligo CHHZ 150 (GTCCTCGTTGTGGGAGGTGATGTC; SEQ ID NO: 48) were used.To detect the UBQ10 promoter oligo CHHZ467 (CTGCCACTCCATTTCCTTCTCGGC;SEQ ID NO: 49) and oligo CHH469 (ACTTATCCGGTCCTAGATCATCAG; SEQ ID NO:50) were used. The results of the PCR analysis are displayed in Table38. TABLE 38 No. of Plants No. of PCR No. of Visual Host No. of EventsTested Positives Positives Broccoli 74 140 127 128 Canola 6 55 35 32Tobacco 24 61 16 20Western Blot

Expression of the fluorescent protein DsRed in the adchromosomalbroccoli plants was measured by Western blot analysis. Leaf tissue,obtained using a cork borer, was ground to a fine powder using a chilledpestle and the leaf tissue was lysed using Novex Tris-Glycine SDS SampleBuffer (2×) on ice. The protein sample concentration was determinedusing the BAC assay and the samples were separated on a tris-glycine gel(4-20%) according to the manufacturer's instructions (Novex). A proteinsample from a non-adchromosomal broccoli plant was run as a negativecontrol. Boiled purified E. coli purified DsRed was run as a positivecontrol. The protein was transferred from the gel to filter paper(nitrocellulose or PVDF). The filter was immunoblotted with ananti-DsRed primary antibody (Clontech), which was detected with anHRP-labeled secondary antibody and developed with Pierce StipersignalWest Pico Chemiluminescent Substrate. Table 39 summarizes the WesternBlot analysis. TABLE 39 Broccoli Centromeres in Soy Centromeres Broccoliin Broccoli No. tested No. positive No. tested No. positive Centromere 99 1 1 Mini- 28 28 1 1 chromosomes Events 51 47 3 3 Plants 136 119 4 4RT-PCR

Expression of the DsRed mRNA in the adchromosomal broccoli plants wasalso analyzed by RT-PCR. Total RNA was isolated from plant tissue usingthe Qiagen mini-kit (catalog no. 74104) according to the manufacturer'sinstructions. The reverse transcriptase reaction was carried out with 11μl DNAase I-treated total RNA, 1 μl oligo-DT (oligo CHR152 specific forDsRed), 1 μldNTP (10 mM each). The reaction was heated to 62° C. for 2minutes and chill on ice, then the following was added: 4 μl 5× 1^(st)stand buffer (Invitrogen), 2 μl DTT (Invitrogen) and 1 μl Superscript IIreverse transcriptase. The mixture was incubated at 42° C. for 1 hour.Subsequently, 80 μl of dH2O was added and the mixture was heatinactivated for 15 minutes at 70° C.

The cDNA samples generated by the reverse transcriptase reaction wereamplified with a PCR reaction carried out with 5 μl cDNA, 2.5 μl 10×Themopol buffer (New England Bioscience), 0.5 μl dNTPs (10 mM each), 1.5μM oligo 1 (20 μM each), 1.5 μl oligo 2 (20 mM each) 13.5 μl H2O and 0.5μl Taq polymerase (New England Bioscience). 83 adchromosomal broccoliplants were tested and 69 were positive for DsRed mRNA expression (73were positive by visual analysis).

Table 40 is a summary of the visual, PCR, Western, and RT-PCR data forthe adchromosomal broccoli plants. TABLE 40 West- Vis- Con- Centro-Event PCR RT ern ual struct mere # Genes # Plants 4 + + + + 5R4-1 BB5 28 6 + + + + 5R4-1 BB51 2 5 7 + + + + 5R4-1 BB5 2 22 10 + + + + 5R4-1 BB52 16 15 + + + + 5R4-1 BB5 2 10 17 + + + + 5R4-1 BB5 2 17 18 + + + +71R1-1 BB71 2 4 23 + + + + 489 integrative 2 1 35 + + + + 816-2 BB5 4 439 − − − − 817-A BB5 4 13 40 + + + + 817-A BB5 4 3 51 + + + + 817-A BB54 7 52 + + + + 819-A BB5 4 4 53 + + + + 819-A BB5 4 3 55 + + + + 819-ABB5 4 6 67 + + + + 824-9 BB5 4 2 69 + + + + 824-9 BB5 4 5 83 − − − −591-1L BB5 3 5 112 + − − − 593-4L BB5 3 5 119 + + + + 593-4L BB5 3 4

EXAMPLE 12 Analysis of Autonomy and Integration of Mini-Chromosomes inAdchromosomal Broccoli Plants

Southern Blot

Southern blot analysis was carried out to analyze whether themini-chromosome was autonomous or integrated into the genome of the ofthe adchromosomal T0 broccoli plants. An autonomous mini-chromosome willhave the same restriction pattern as wild type plant DNA spiked withmini-chromosome DNA, while a mini-chromosome that has integrated into ahost chromosome will have an altered restriction pattern and thataltered restriction pattern is not predictable. If integration doesoccur and the junction between the host chromosome DN is in thecentromeric region of the mini-chromosome, a restriction pattern similarto an autonomous mini-chromosome is expected. This is true because onlythe “gene region” (the part of the mini-chromosome excluding thecentromere region) is subsequently used as a probe, as described below.

Plant tissue from the adchromosomal and control broccoli plants wasground to a fine powder in liquid nitrogen using mortar and pestle.Genomic DNA was isolated from the homogenized plant cells usingphenol:chloroform:isoamyl alcohol extraction as taught by Csail et al.,(Plant Mol. Biol. Rep. 16: 69-89, 1998). The DNA samples (5 μg) weredigested with BglII restriction enzyme diluted in enzyme buffer, 100×BSA, 100 mM P-mercaptoethanol, 100 mM spermidine, dH₂O. DNA was digestedovernight at 37° C. Subsequently, an additional 2 μl of BglII added tothe DNA and allowed to digest a few additional hours. Loading buffer wasadded and the samples were separated on a 0.7% agarose gel. The DNA onthe gel was visualized using ethidium bromide. The DNA on the gel wastransferred to a nylon membrane using a Bio-Rad Vacuum Blotter (Model785). The filters were probed with radiolabeled DNA complementary to thegene region of the mini-chromosome (entire sequence excluding thecentromere region).

Southern blot analysis was carried out on 60 adchromosomal broccoliplants in which 32 events were tested, 7 centromeres were tested (6broccoli centromeres and 1 soybean centromere). 40% of the samples weretested in duplicate. The Southern Blot results are summarized in Table41. In the majority of events, the gene region of the mini-chromosomewas intact and the results indicate the mini-chromosomes were autonomousor integrated via the centromere sequence. BB5R4-1 TABLE 41 No. of Mini-Events Consistent with Not chromosome Centromere Tested AutonomyIntegration Detected 5R4-1 BB5 6 1 5 — 5R16-6 BB5 1 1 — — 817 BB5R4-1 21 — 1 818 BB5R4-1 1 1 — — 819 BB5R4-1 4 4 — — 820 BB5R4-1 1 — — 1 823BB5R4-1 3 3 — — 824 BB5R4-1 2 1 — 1 591-1 BB5R4-1 2 1 — 1 593-3L BB5R4-11 1 — — 593-4L BB5R4-1 1 — 1 — 816-2 BB5R4-1 2 2 — — 965 BB15R4-1 1 1 —— 964-4 BB15R4-1 1 — — 1 967 BB16R1-2 1 1 — — 222R2-1 BB222 1 — — 1972-5 BB60R1-1 1 1 — — SB986-1 SB38R2-2 1 1 — — Total 32 20  6 6Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) is carried out to determinethe autonomy of the mini-chromosomes in root tips and anthers from theadchromosomal T0 broccoli plants. The tissues were probed with labeledpCHR151 and pCHR08, and BB5 PCRed BSAT and stained with DAPI. Foranalysis, the FISH chromosomal spreads needed to meet the followingcriteria: condensed and well-spread chromosomes, free of majorbackground, strong centromere hybridization, 18 chromosomes, signallocalized to approximately the same place on same chromosome.Integration is determined by detecting the mini-chromosome and it isassociated with the genome and autonomy is determined by detecting themini-chromosome and it was free of the genome. If the mini-chromosome isdetected to be both free and associated with the genome it is bothautonomous and integrated mini-chromosomes are present.

EXAMPLE 13 Analysis of Mini-chromosomes in T1 Brassica Pollen and T1Brassica Plants

To analyze the presence of the mini-chromosome in pollen isolated from aflowering adchromosomal T0 Brassica plant (T1 pollen), two anthers fromeach of three flowers were removed. The anthers were harvested fromflowers that were open for more than a half a day and were sheddingpollen. The anthers were streaked on a plate of sterile mediumcontaining 1× MS salts, 13% sucrose, 0.8% tissue culture agar, pH 5.8,depositing a streak of pollen onto the surface of the plate. In adarkened room, the pollen was examined with a fluorescencestereo-microscope using 100× magnification and a rhodamine or FITCfilter set. At least 500 pollen cells in groups of 100 were counted.Total pollen cells were counted under visible light and then examinedunder fluorescence.

T1 pollen was analyzed from adchromosomal T0 broccoli plants. Thebroccoli pollen visual data is summarized in Table 42. TABLE 42 % ofPollen Grains Plant Event Expressing DsRed Pbo4A 4 0% pbo4E2 4 no pollenpbo7BCopy 7 0% pbo7C 7 no pollen pbo7C1Copy 7 no pollen pbo7S 7 0%pbo10C2 10 no pollen pbo10C2Copy 10 no pollen pbo10D3 10 no pollenpbo15E1 15 0% pbo15E2 15 7% pbo15M1 15 1% pbo15O 15 0% pbo17A1 17 0%pbo17C2 17 0% pbo17G1 17 0% pbo17N1 17 0% pbo18A 18 1% pbo18B 18 0%pbo19G3 19 4% pbo28A 28 0% pbo39B1 39 0% pbo39C 39 0% pbo40A 40 32% pbo40D 40 25%  pbo51C 51 4% pbo51D 51 0% pbo52A 52 0% pbo52C 52 1%pbo53A 53 8% pbo55B 55 4% pbo69A1 69 0% pbo83A1 83 0% pbo112A3 112 nopollen pbo119A 119 no pollen pbo126A 126 0% pbo126B 126 3% pbo173D 173no pollen pbo221A 221 14%  pbo222E 222 2%

Adchromosomal T1 broccoli plants were generated by selfing oroutcrossing. All crosses were done by bud pollination to overcomeself-incompatibility and/or to ensure that the donor pollen gave rise toall seeds in the pod. To perform a bud pollination, an unopened flowerwas stripped of all sepals, petals and stamens, leaving only theimmature pistil. Pollen from the appropriate plant was applied to thestigma. The flower was labeled and the pod allowed to develop normallyThe presence of the mini-chromosome in the adchromosomal T1 broccoliplants were analyzed visually and by PCR as described in Example 8.Three mini-chromosomes comprising one of two centromere sequences (with2 and 4 genes) were analyzed. The data from the adchromosomal T1broccoli plants is summarized in Table 43 and Table 44. TABLE 43 T1Adchromosomal_Broccoli Plants Visual Visual Visual scoring of scoring ofScoring T1 seedlings T1 seedlings of T1 PCR on (outcross - (outcross -seedlings negative male) female) (self) seedlings Event tested positivetested positive tested positive tested positive 4 68 0 55 0 — — 57 0 17158 0 — — 1 0 95 0 17 70 0 — — 1 0 — — 17 39 0 67 0 17  1 — — 17 9 0  40 — — 13 1 18 11 0 52 5 — — — — 18 34 0 — — — — — — 40 46 0  4 3 9 7  30

TABLE 44 T1 Adchromosomal Broccoli Plants Visual PCR of Donor RecipientSelf Negatives Plant Event Tested Positive Tested Positive TestedPositive Tested Positive pbo4A 4 69 0 55 0 0 0 57 0 pbo17A1 17 258 0 510 9 0 98 0 pbo17A3 17 77 0 1 0 1 0 0 0 pbo17C2 17 43 0 65 0 20 0 0 0pbo17G1 17 0 0 4 1 0 0 0 0 pbo17N1 17 9 0 4 0 10 0 13 0 pbo18A 18 248 052 0 6 0 0 0 pbo18B 18 50 0 29 0 0 0 0 0 pbo22 22 66 0 0 0 0 0 0 0pbo40A 40 56 28 3 1 9 7 59 27 pbo40D 40 65 32 22 13 40 31 0 0 pbo51A 5164 0 0 0 12 0 0 0 pbo51C 51 101 0 63 2 0 0 0 0 pbo51D 51 0 0 0 0 2 0 0 0pbo52C 52 83 0 236 6 59 0 0 0

Pollen was also analyzed from transformed T0 canola plants (Brassicanapus) by visual analysis. The visual data is summarized in Table 45.TABLE 45 Brassica napus pollen fluorescence summary % Event # pollenfluorescent Plant # Mini-C counted pollen Flower size 11-1-2 11pCHR820-1 568 87.5% large 11-1-4 11 pCHR820-1 558 88.9% large 11-2 11pCHR820-1 637 68.8% N.R. 11-3-1-1 11 pCHR820-1 613 52.7% small 11-3-1-211 pCHR820-1 667 63.7% small 11-6-1 11 pCHR820-1 591 98.0% large 11-6-211 pCHR820-1 580 96.6% large 11-6-3 11 pCHR820-1 640 93.8% large 11-6-411 pCHR820-1 585 95.2% large 11-7 11 pCHR820-1 607 67.2% small 11-8-1-111 pCHR820-1 540 65.4% N.R. 11-8-1-2 11 pCHR820-1 584 62.5% small11-8-1-3 11 pCHR820-1 512 64.8% N.R. 11-8-2 11 pCHR820-1 542 66.4% N.R.11-8-2-2 11 pCHR820-1 652 69.8% small 11-8-2-3 11 pCHR820-1 574 66.7%small 11-8-2-4 11 pCHR820-1 610 63.1% small 11-12 11 pCHR820-1 550 94.2%N.R. 11-14-1-1 11 pCHR820-1 600 97.3% large 11-14-2 11 pCHR820-1 56997.7% large 11-15 11 pCHR820-1 565 86.7% large 11-17 11 pCHR820-1 60598.0% large 16-2-2 16 pCHR820-1 >2000 0.0% large/abnormal 16-2-3 16pCHR820-1 655 61.1% large/abnormal 16-2-3 16 pCHR820-1 589 48.9%large/abnormal 19-2 19 pCHR820-1 573 40.1% large 19-3-1 19 pCHR820-1 70449.7% large 19-5-1 19 pCHR820-1 666 49.7% LargeN.R. = not recorded

Pollen was also analyzed from adchromosomal T0 tobacco plants by visualanalysis. The visual pollen data is summarized in Tables 46. Inaddition, adchromosomal T1 tobacco plants were analyzed visually and byPCR. The T1 plant data is summarized in Tables 47 and 48. T1 tomatopollen, harvested from adchromosomal T0 tomato plants was also visuallyanalyzed. 537 pollen cells were counted and 153 were fluorescent (28.5%)TABLE 46 Visual Analysis of Adchromosomal T0 Tobacco Plants ConstructFluorescent Total Plant type Construct pollen pollen % XNN — — 0 2000 0pNt1E mini-C TB99R7-1 79 511 15.5 pNt2A-3 mini-C TB99R10-1 289 531 54.4pNt2D-1 mini-C TB99R10-1 323 538 60.0 pNt2H-2 mini-C TB99R10-1 251 52048.0 pNt2E mini-C TB99R10-1 268 507 53.0 pNt2D mini-C TB99R10-1 195 50838.0 pNt2H mini-C TB99R10-1 76 507 15.0 pNt4D-1 mini-C TB99R10-1 200 50239.8 pNt6A mini-C pCHR488 + 18 120 15.0 TB99R1-5 pNt8B integratingpCHR488 + 479 63 650 9.7 pNt13D integrating pCHR488 + 480 188 519 36.2pNt15B mini-C TB99R8-1 249 500 49.8 pNt15D mini-C TB99R8-1 272 506 54.0pNt16B mini-C TB132R8-1 202 501 40.0 pNt16D mini-C TB132R8-1 277 50655.0

TABLE 47 Visual Analysis of Adchromosomal T0 Tobacco Plants PCR scoringT0 Visual Scroing of T1 of red (+) T1 PCR scoring of Plant seedlings(self) seedlings non-Red (−) T1 Visual % (selfed) seedlings (self) EventScore counted Red Red tested positive tested positive 1 + 192 174 90% 106 16 0 1 + 91 79 87% 2 − 294 0 0% 2 − 393 1 0% 2 + 208 163 78% 10 10 250 2 + 177 126 71% 2 + 200 145 73%

TABLE 48 Visual Analysis of T1 Tobacco Seedlings Fluorescent SeedlingsPlant Seedlings Counted % Fluorescent pNT 14B 0 165   0% pNT 15B 36 6162.30%  pNT 15D 137 210 62.00%  pNT 1B(1) 79 91 86.80%  pNT 1G(2) 174192   90% pNT 2A-1 163 208   78% pNT 2A-2 65 89 73.00%  pNT 2A-3(2) 145200 72.5% pNT 2B-1 0 73   0% pNT 2C(2) 1 393 0.25% pNT 2C-1 0 294   0%pNT 2C-2(1) 0 204   0% pNT 2D(1) 126 177 71.1% pNT 2D(3) pNT 2E(1) 185238 77.7% pNT 2F(1) 0 228   0% pNT 2F(2) pNT 2G(1) 14 19 73.7% pNT 2G(2)16 24 66.7% pNT 2I(1) 0 230   0% pNT 2K(2) 156 226 69.00%  pNT 2D(2) pNT5A 0 128 0 pNT 15A 0 78 0 pNT 19A 0 217 0

EXAMPLE 14 Corn Centromere Discovery

BAC Library Construction

Two Bacterial Artificial Chromosome (BAC) libraries were constructedfrom corn genomic DNA. The corn genomic DNA was isolated from cornvariety B73 and digested with the restriction enzymes BstYI or MboI.These enzymes were chosen because they are methylation insensitive andtherefore can be used to enrich BAC libraries for centromere DNAsequences.

Probe Identification and Selection

Twenty-three groups of corn repetitive genomic or plastid sequences,including specific centromere-localized sequences, were initiallycompiled as candidate probes for hybridization with the BAC libraries(Table 49). These probes represented various classes of corn repetitivesequences including satellite repeats(heterochromatic/centromere-specific), retroelements, rDNA, Bchromosome-specific repeats, chloroplast and mitochondrion DNA,hypermethylated or hypomethylated DNA fractions, and telomeric DNA.TABLE 49 Maize Repetitive Sequences and Bac Library Probes GenBank ClassClass Name Primers Description Reference Comment accession 1 CR CRJM-001gypsy-type Aragon- aka CRM, AY1290008 (centromeric and 002 localized toAlcaide et al pSau3A9 retrotransposable) cen of all 1996, Jiang et (fromelement cereals. CentC al 1996, Zhong sorghum), and CRM co- et al 2002CRR (from IP with CEN rice) H3 2 Cent-A CHR 15 and centromere AF082532AF078917 16 retrotransposon, Similar includes sequence MCS1A and B 3Huck CRJM-005 Ty3/gypsy Meyers et al (most AF050438 and 006 2001frequent) 4 Grande CRJM-056 Ty3/gypsy Meyers et al AF050437 and 057 20015 Cinful CRJM-007 Ty3/gypsy Meyers et al AF049110 and 008 2001 6Ji/Prem2 LTR-5 Ty1/copia Meyers et al from alpha CRJM-011 2001 zein seqand 012 gag CRJM- 013 and 014 7 Opie Ty1/copia Meyers et al 5′ LTRAF050453 2001 8 Tekay CRJM-009 3′ LTR AF050452 and 010 9 alpha zeinAF090447 10 adh AF123535 11 bz AF448416 12 knob 180 CHR 11 and manygi|168710|gb| 12 sequences! M32521.1| MZEZMA 13 MZEHETRO CRJM-015 maizePeacock et al M35408 and 016 heterochromatic PNAS. 78, repeat (knob)4490-4494 (1981) 14 TR-1(knob CHR 13 and Knob-specific Hsu et al 2002 3lengths, AF071126 360) 14 multi types. Type 1 BLASTs to all 3. Cuts w/RI15 Cent-C CHR 17 and 156 bp Ananiev et al all match AY321491 18 1998well (Cent C27) — AF078923 158a CRJM-019 AF078922 and 020 156a 16 Cent4CRJM-021 Chromosome 4 Page et al, AF242891 and 022 repeat 2001homologous to B- chromosome cen repeat 17 pZmBs and S67586 B-specificAlfenito and AY173950 K5 repeats; B73 Birchler 1993; has no B Kaszas andchromosomes Birchler 1993, 1998 18 rDNA CRJM-023 maize AF013103 and 024intergenic spacer CRJM-025 maize 5S AF273104 and 026 CRJM-027 maize 17SK0220 and 028 19 chloroplast CHHZ211 Arabidiosis and 212 CRJM-030 maizexpl X01365 and 031 rDNAs 20 mito CHHZ214 Arabidiosis and 215 CRJM-032maize mito K01868 and 033 26S rDNA 21 hypermethylated purified complexfraction mixture 22 hypomethylated purified complex fraction mixture 23telomere sub-telomeric U39641 U39642 repeat

Twelve probes were picked to interrogate the BAC libraries. These probesrepresent different groups of commonly found repetitive sequences in thecorn genome. The twelve probes selected are shown in Table 49 and 50 andwere: Cent-C (#15 ), Cent4 (#16), MZEHETRO (#13), TR-1 (#14), CentA(#2), CR (#1), Huck #3), Grande (#4), 17S rDNA (#18), 5S rDNA (#18); Bcen (#17), and xplmito (#19 and #20). The primers used to amplify theseprobes are identified in Table 49. Probes were prepared and labeled withstandard molecular methods. TABLE 50 Class Cent- 17S 5S # clones ClassProperties C Cent-A CR Huck Grande rDNA Cent4 TR-1 MZEHETRO rDNA B cenxplmito identified I HiC LoA >=7 <7 <7 <7 <6 N/A N/A N/A N/A N/A N/A N/A61 II HiC HiA >=7 >=6 <7 <=10 <=10 N/A N/A N/A N/A N/A N/A N/A 61 IIIHiCR HiC >=7 <6 >=6 <=10 <=10 N/A N/A N/A N/A N/A N/A N/A 30 IV HiAHiC >=7 >6 >=6 <=10 <=10 N/A N/A N/A N/A N/A N/A N/A 30 HiCR V HiCHi17s >=7 >0 >0 >0 >0 >5 N/A N/A N/A N/A N/A N/A 30 VI Hi4 >0 >0 >0 >0N/A N/A >5 N/A N/A N/A N/A N/A 17 VII HiTr1 >0 >0 N/A N/A N/A >0 N/A >6<6 N/A N/A N/A 31 LoHet VIII LoTr1 >0 >0 N/A N/A N/A >0 N/A <5 >7 N/AN/A N/A 31 HiHet IX HiTr1 >0 >0 N/A N/A N/A >0 N/A >6 >6 N/A N/A N/A 24HiHet Total 315*Values represent hybridization intensities of an individual BAC to eachprobe on a scale of 1 to 10. Values were normalized.Library Interrogation and Data Analysis

The BAC clones from the libraries were spotted onto filters for furtheranalysis. The filters were hybridized with each of the 12 probes toidentify specific BAC clones that contain DNA from the group ofsequences represented by the probe(s).

A total of 92,160 BAC clones from the two libraries (36,864 BAC clonesfrom 2 filters from the BstYI library and 55,296 clones from 3 filtersfrom the MboI library) were interrogated with each of the 12 probesdescribed above sing the following hybridization conditions: 0.5×SSC0.25% SDS at 65 degrees for 15 minutes, followed by a wash at 65 degreesfor a half hour. The hybridization intensities of the BAC clones witheach probe were scanned to quantitate hybridization intensity for eachclone. Scores of 1 to 10 (based on the hybridization intensities, with10 being the strongest hybridization) were imported into a relationaldatabase, for classification. The database contained a total of 24tables, 12 from each library used in the interrogation. Each tablecontained the hybridization scores of each BAC clone from the BstYI orMboI library, to one of the 12 probes. Data analysis was carried outusing standard SQL (Structured Query Language) routines to find BACsthat contain different groups of repetitive sequences.

Classification and Selection of BAC Clones for Mini-ChromosomeConstruction

BAC clones containing centromeric/heterochromatic DNA were identified bytheir hybridization scores to different probes. The goal was to selectBAC clones that contained a diverse set of various repetitive sequences.Nine classes of centromeric BAC clones were eventually chosen to coverthe broadest possible range of centromeric/heterochromatic sequences formini-chromosome construction. Detailed descriptions of each class andprobe hybridization values for each class are shown in Table 50.

Class I (HiC LoA) BAC clones had strong hybridization to probe Cent-C,but low hybridization to Cent-A, CR, Huck and Grande. Class II (HiC HiA)BAC clones had strong hybridization to both Cent-C and CentA, but lowhybridization to CR. Class III (HiCR HiC) BAC clones had stronghybridization to both Cent-C and CR, but low hybridization to CentA.Class IV (HiA HiC HiCR) BAC clones had strong hybridization to Cent-C,CentA, and CR. Class V (HiC Hi17s) BAC clones had strong hybridizationto Cent-C and 17S rDNA. Class VI (Hi4) BAC clones had stronghybridization to Cent4. Class VII (HiTr1 LoHet) BAC clones had stronghybridization to TR-1 but low hybridization to MZEHETRO. Class VIII(LoTr1 HiHet) BAC clones had strong hybridization to MZEHETRO but lowhybridization to TR-1. Class IX (HiTr1 HiHet) BAC clones had stronghybridization to both TR-1 and MZEHETRO.

A number of representative clones from each class were chosen to yield atotal of 315 BAC clones for further analysis by restriction digestfingerprinting.

The 315 BAC clones were fingerprinted based on restriction sites foundin the centromere specific sequence(s). Fingerprinting was used toevaluate the sequence composition of the large numbers of BAC clones andto compare their similarity to each other by comparing the restrictionenzyme digest fragment patterns. A sequence with a tandem repeatedsequence will show a single intense band of unit repeat size whendigested with a restriction enzyme that cuts within the unit repeat.Second, BAC clones with similar sequences will show similar patterns ofrestriction fragments in a digest.

BAC DNA was extracted from bacteria using methods familiar to those inthe art. Restriction enzymes HpaII and MspI were used to digest BACclones in Classes I through VI, and restriction enzyme NdeI was used todigest BAC clones in classes VII through IX.

Z. mays (corn) BACs ZB19 and ZB113 were deposited with the American TypeCulture Collection (ATCC) P.O. Box 1549 Manassas, Va. 20108, USA on Feb.23, 2005 and assigned Accession NO. ______.

EXAMPLE 15 Construction of Maize Mini-Chromosomes

The 115 BAC clones identified in Example 1 were grown up and DNA wasextracted for mini-chromosome construction using NucleoBond™Purification Kit (Clontech). To determine the molecular weight ofcentromere fragments in the BAC libraries, a frozen sample of bacteriaharboring a BAC clone was grown in selective liquid media and the BACDNA harvested using a standard alkaline lysis method. The recovered BACDNA was restriction digested and resolved on an agarose gel. Centromerefragment size was determined by comparing to a molecular weightstandard.

For each BAC, two types of mini-chromosomes were generated, differingonly by the promoter used to express the DsRed gene. Corn ADH promoterwas used to express DsRed in mini-chromosomes constructed with pCHR667and the Arabidopsis UBQ10 promoter was used to express DsRed inmini-chromosomes constructed with pCHR758. Mini-chromosome geneticelements within the pCHR667 and pCHR758 vectors are set out in Table 51and 52, respectively. TABLE 51 Donor Components of pCHR667 Size GeneticElement (base pair) Location (bp) Details ADH Corn Promoter 1189 14-1202 PCR amplified maize promoter alcohol dehydrogenase 1 (ADH-1)for expression of DsRed in maize (used primers CRJM-42/43) Maize ADHIntron 579 1216-1794 PCR amplified maize ADH intron with AUG mutationfor stabilization of DsRed2 gene transcript and increase proteinexpression level (used primers CRJM-72/73) DsRed2 + NLS 780 1817-2596Nuclear localized red fluorescent protein from Discosoma sp. (Matz, Met. al Nat Biotechnol 1999 Dec; 17(12): 1227). ADH Terminator 2032725-2927 Amplified maize terminator using primers CRJM-46/47 BacterialKanamycin 817 3066-3882 Bacterial kanamycin selectable marker Rps16Aterminator 489 4065-4553 Amplified from Arabidopsis thaliana 40Sribosomal protein S16 (At2g09990) for termination of NptII gene NPTII795 4617-5411 Neomycin phosphotransferase II plant selectable markerUBQ10 intron 359 5439-5798 PCR amplified Arabidopsis thaliana intronfrom UBQ10 gene (At4g05320) for stabilization of NptII gene transcriptand increase protein expression level YAT1 yeast promoter 2000 5812-7811PCR amplified YAT1 promoter from chromosome I of Saccharomycescerevisiae for expression of NptII in maize LoxP 34 10341-10374 andRecombination site for Cre 7829-7862 mediated recombination (Arenski et.al 1983, Abremski et. al 1984)

TABLE 52 Donor Components of pCHR758 Size Genetic Element (base pair)Location (bp) Details UBQ10 promoter 2038  14-2051 Arabidopsis thalianapolyubiquitin promoter (At4g05320) DsRed2 + NLS 780 2088-2867 Nuclearlocalized red fluorescent protein from Discosoma sp. (Matz, M et. al NatBiotechnol 1999 Dec; 17(12): 1227). Pyruvate kinase 332 3002-3333Arabidopsis thaliana pyruvate terminator kinase terminator (At5g52920)Bacterial Kanamycin 817 3478-4294 Bacterial kanamycin selectable markerRps16A terminator 489 4477-4965 Amplified from Arabidopsis thaliana 40Sribosomal protein S16 (At2g09990) for termination of NptII gene NPTII795 5029-5823 Neomycin phosphotransferase II plant selectable markerUBQ10 intron 359 5851-6210 PCR amplified Arabidopsis thaliana intronfrom UBQ10 gene (At4g05320) for stabilization of NptII gene transcriptand increase protein expression level YAT1 yeast promoter 2000 6224-8223PCR amplified YAT1 promoter from chromosome I of Saccharomycescerevisiae for expression of NptII in maize LoxP 34 8243-8276 &Recombination site for Cre 10755-10788 mediated recombination (Arenskiet. al 1983, Abremski et. al 1984)

Corn mini-chromosomes were constructed by following a two-stepprocedure: Step 1: Preparation of donor DNA for retrofitting with BACcentromere vectors and Step 2: Cre-Lox Recombination-BAC and Donor DNAto generate the mini-chromosome. A total of 230 corn mini-chromosomeswere constructed using this assembly process, and were subsequentlytested in several different corn cell lines.

Preparation of Donor DNA for Retrofitting

Cre recombinase-mediated exchange was used to construct mini-chromosomesby combining the plant centromere fragments cloned in pBeloBAC111 with adonor plasmid (i.e. pCHR667 or pCHR758, tables 51 & 52). The recipientBAC vector carrying the plant centromere fragment contained a loxPrecombination site; the donor plasmid contained two such sites, flankingthe sequences to be inserted into the recipient BAC.

Cre recombinase-mediated exchange was used to construct mini-chromosomesby combining the plant centromere fragments cloned in pBeloBAC11 with adonor plasmid (i.e. pCHR667 & pCHR758, Tables 7 and 8). The recipientBAC vector carrying the plant centromere fragment contained a loxPrecombination site; the donor plasmid contained two such sites, flankingthe sequences to be inserted into the recipient BAC. Mini-chromosomeswere constructed using a two-step method. First, the donor plasmid waslinearized to allow free contact between the two loxP site; in this stepthe backbone of the donor plasmid is eliminated. In the second step, thedonor molecules were combined with centromere BACs and were treated withCre recombinase, generating circular mini-chromosomes with all thecomponents of the donor and recipient DNA. Mini-chromosomes weredelivered into E. coli and selected on medium containing kanamycin andchloramphenicol. Only vectors that successfully cre recombined andcontained both selectable markers survived in the medium.Mini-chromosomes were extracted from bacteria and restriction digestedto verify DNA composition and calculate centromere insert size.

To determine the molecular weight of the centromere fragments in themini-chromosomes, three bacterial colonies from each transformationevent were independently grown in selective liquid media and themini-chromosome DNA harvested using a standard alkaline lysis method.The recovered mini-chromosome was restriction digested and resolved onan agarose gel. Centromere fragment size was determined by comparing toa molecular weight standard. If variation in centromere size was noted,the mini-chromosome with the largest centromere insert was used forfurther experimentation.

Functional Testing of Mini-Chromosomes Using Transient Assays

Maize mini-chromosomes were tested in several corn cell lines includingPC1117, HiII, and BMS, and the procedure was optimized for antibioticselection, cell pre-treatments, and bombardment conditions. All assayswere transient and fluorescent cells were counted at several timepoints. Preliminary results identified several mini-chromosomes thatsuccessfully generated fluorescent cell clusters.

EXAMPLE 16 Transformation and Selection of Regenerable Cells and CornPlant Regeneration

The biolistic delivery method using wet gold particles, described inExample 2, was used to deliver the mini-chromosomes into a number ofdifferent corn tissues including suspension cells, plate-grown calli,and immature embryos. For the purpose of transient delivery or selectionof stable cell culture modified with a corn mini-chromosome, suspensioncells were used for delivery using wet or dry gold delivery methods. Anexample of such a suspension culture is the publicly available line,PC1117.

To obtain trans-chromosomal corn plants modified with cornmini-chromosomes, standard protocols for corn tissue culture andtransformation are followed. Such protocols include the MaizeEmbryo/Callus Bombardment Protocols available at Iowa Statue University,College of Agriculture web site.

The transformation process involved the preparation of regenerabletissues such as immature embryos from corn cultivars such as Hill,pre-culture of embryos on an auxin-enriched medium, delivery of miniC'sinto immature embryos or embryogenic calli, selection and isolation offluorescent cell clusters, expansion of cell clusters and formation oftranschromosomal embryos, maturation and regeneration of embryos intowhole plants.

1. An adchromosomal plant comprising a mini-chromosome, wherein saidmini-chromosome has a transmission efficiency during mitotic division ofat least 90%.
 2. The plant according to claim 1, wherein themini-chromosome has a transmission efficiency during mitotic division ofat least 95%.
 3. The plant according to claim 1 or 2, wherein themini-chromosome has a transmission efficiency during meiotic division ofat least 80%.
 4. The plant according to claim 3, wherein themini-chromosome has a transmission efficiency during the meioticdivision of at least 85%.
 5. The plant according to claim 4, wherein themini-chromosome has a transmission efficiency during meiotic division ofat least 90%.
 6. The plant according to claim 5, wherein themini-chromosome has a transmission efficiency during meiotic division ofat least 95%.
 7. The plant according to to any one of claims 1-6,wherein the mini-chromosome is 1000 kilobases or less in length. 8 Theplant according to claim 7 wherein the mini-chromosome is 600 kilobasesor less in length. 9 The plant according to claim 8 wherein themini-chromosome is 500 kilobases or less in length.
 10. The plantaccording to any one of claims 1-9, wherein the mini-chromosomecomprises a site for site-specific recombination.
 11. The plantaccording to any one of claims 1-10, wherein the mini-chromosomecomprises a centromeric nucleic acid insert derived from a crop plantcentromere.
 12. The plant accoring to claim 12, wherein the cetromericnucleic acid insert is derived from genomic DNA of a plant selected fromthe group consisting of Brassica, Nicotiana, Lycopersicum, Glycine, orZea species.
 13. The plant according to claim 12, wherein thecentromeric nucleic acid insert is derived from genomic DBA of a plantseleced from the group consisting of broccoli, canola, tobacco, tomato,soybean or corn.
 14. The plant according to to any one of claims 1-13,wherein the mini-chromosome comprises a centromeric nucleic acid insertthat comprises artificially synthesized repeated nucleotide sequences.15. The plant according to any one of claims 1-14, wherein themini-chromosome is derived from a donor clone or a centromere clone andhas substitutions, deletions, insertions, duplications or arrangementsof one or more nucleotides in the mini-chromosome compared to thenucleotide sequence of the donor clone or centromere clone.
 16. Theplant of claims 1-15, wherein the mini-chromosome is obtained by passageof the mini-chromosome through one or more hosts.
 17. The plant of claim16, wherein the mini chromosome is obtained by passage of themini-chromosome through two or more different host.
 18. The plant ofclaim 17, wherein the host is selected from the group consisting ofviruses, bacteria, yeast, plants, prokaryotic organisms, or eukaryoticorganisms.
 19. The plant according to any one of claims 1-18 wherein themini-chromosome comprises one or more exogenous nucleic acids.
 20. Theplant according to claim 19, wherein the mini-chromosome comprise atleast two or more exogenous nucleic acids.
 21. The plant according toclaim 20, wherein the mini-chromosome comprise at least two or moreexogenous nucleic acids.
 22. The plant according to claim 21, whereinthe mini-chromosome comprise at least two or more exogenous nucleicacids.
 23. The plant according to claim 22, wherein the mini-chromosomecomprise at least two or more exogenous nucleic acids.
 24. The plantaccording to claim 19, wherein the mini-chromosome comprise at least twoor more exogenous nucleic acids.
 25. The plant according to claims19-24, wherein at least one exogenous nucleic acid is operably linked toa heterologous regulatory sequence functional in plant cells.
 26. Theplant according to claim 25, wherein the regulatory sequence is a plantregulatory sequence.
 27. The plant according to claim 25, wherein theregulatory sequence is a non-plant regulatory sequence.
 28. The plantaccording to claim 27, wherein the regulatory sequence is an insect oryeast regulatory sequence.
 29. The plant according to claim 27, whereinthe non-plant regulatory sequence comprises any on of the SEQ ID NOS: 4to 23 or a functional fragment thereof.
 30. The plant according to anyone of claims 1-29, wherein the the mini-chromosome comprises anexogenous nucleic acid that confers herbicide resistance, insectresistance, disease resistance, or stress resistance on the plant. 31.The plant according to claim 30 wherein the exogenous nucleic acidconfers resistance to phosphinothricin or glyphosate herbicide.
 32. Theplant according to claim 31 wherein the exogenous nucleic acid encodes aphosphinothricin acetyltransferase or a mutant enoylpyruvly shikimatephospate (EPSP) synthase.
 33. The plant according to any one of clamis1-32, wherein the mini-chromosome comprises an exogenous nucleic acidthe encodes a Bacillus thuringiensis crystal toxin gene or Bacilluscereus toxin gene.
 34. The plant according to anyone of claims 1-38,wherein the mini-chromosome comprises an exogenous nucleic acid thatconfers resistance to drought, heat, chilling, freezing, excessivemoisture, ultraviolet light, ionizing radiation, mechanical stress,toxins, pollution, or salt stress.
 35. The plant according to any one ofclaims 1-34, wherein the mini-chromosome comprises an exogenous nucleicacid that confers resistance to a virus, bacteria, fungi or nematode.36. The plant according to any one of claims 1-35, wherein themini-chromosome comprises an exogenous nucleic acid conferring herbicideresistance, an exogenous nucleic acid conferring insect resistance, andat least one additional exogenous nucleic acid.
 37. The plant accordingto any one of claims 1-36, wherein the mini-chromosome comprise anexogenous nucleic acid is selected from the group consisting of anitrogen fixation gene, a plant stress induced gene, a nutrientutilization gene, a gene encoding a secretable antigen, a toxin gene, areceptor gene, a ligand gene, a seed storage gene, a hormone gene, anenzyme gene, an interleukin gene, a clothing factor gene, a cytokinegene, an antibody gene, a growth factor gene, a transcription factorgene, a transcriptional repressor gene, a DNA-binding protein gene, arecombination gene, a DNA replication gene, a programmed cell deathgene, a phosphatase gene, a G protein gene, a cyclin gene, a cell cyclecontrol gene, a gene involved in transcription, a gene involved intranslation, a gene involved in RNA processing, a gene involved In RNAi,an organellar gene, a intracellular trafficking gene, an integralmembrane protein gene, a transporter gene, a membrane channel proteingene, a cell wall gene, a gene involved in protein processing, a geneinvolved on protein modification, a gene involved on proteindegradation, a gene involved in metabolism, a gene involved onbiosynthesis, a gene in assimilation of nitrogen or other elements ornutrients, a gene involved in controlling carbon flux, gene involved inrespiration, a gene ibnvolved in photosynthesis, a gene involved inlight sensing, a gene involved in organogenesis, a gene involved inembryogenesis, a gene involved in differentiation, a gene involved inmeiotic drive, a gene involved in self incompatibility, a gene involvedin development, a gene involved in nutrient, metabolite or mineraltransport, a gene involved in nutrient, metabolite storage, acalcium-binding protein gene, or lipid-binding protein gene.
 38. Theplant according to claim 37, wherein the enzyme gene is selected fromthe group consisting of a gene that encodes an enzyme involved inmetabolizing biochemical waste for use in bioremdation, a gene thatencodes an enzyme for modifying pathways that produce secondary plantmetabolites a gene that encodes an enzyme that produces apharmaceutical, a gene that encodes an enzyme that improves changes thenutritional content of a plant, a gene that encodes an enzyme involvedin vitamin synthesis, a gene that encodes an enzyme involved incarbohydrate, polysaccharide or starch synthesis, a gene that encodes anenzyme gene that encodes an enzyme involved in fatty acid, fat or oilsynthesis, a gene that encodes an enzyme involved in synthesis of a fueland a gene that encodes an enzyme involved in synthesis of a fragrance,a gene that encodes an enzyme involved in synthesis of a flavor, a genethat encodes an enzyme involved in synthesis of a structural or fibrouscompound, a gene that encodes an enzyme involved in synthesis of a foodadditive, a gene that encodes an enzyme involved in synthesis of achemical insecticide, a gene that encodes an enzyme involved insynthesis of an insect repellent, or a gene controlling carbon flux in aplant.
 39. The plant according to any one of claims 37, wherein the thecentromere of the mini-chromosome comprises n copies of a repeatednucleotide sequence, wherein n is less than
 1000. 40. The plantaccording to any one of claims 1-38, wherein the centromere of themini-chromosome comprises n copies of a repeated nucleotide sequence,wherein n is at least
 5. 41. The plant according to any one of claims1-38, wherein the centromere of the mini-chromosome comprises n copiesof a repeated nucleotide sequence, wherein n is at least
 15. 42. Theplant according to claim 41, wherein the centromere of themini-chromosome comprises n copies of a repeated nucleotide sequence,wherein n is at least
 50. 43. The plant according to anyone of claims1-42 wherein the mini-chromosome comprises a telomere.
 44. The plantaccording to anyone of claims 1-42, wherein the mini-chromosome iscircular.
 45. The plant according to any one of claims 1-44, wherein theplant is a monocotyledone.
 46. The plant according to any one of claims1-44, wherein the plant is a dicotyledone.
 47. The plant according toany one claims 1-44, wherein the plant is a cereal plant.
 48. The plantaccorsing to any one of claims 1-44, wherein the plant is from theBrassica, Nicotiana, Lycopersicum, Glycine or Zea species.
 49. The plantaccording to any one of claims 1-44, wherein the plant is a vegatablecrop.
 50. The plant accroding to any one of claims 1-44, wherein theplant is a field crop.
 51. The plant according to any one of claims1-44, where the plant is a fruit and vine crop.
 52. The plant accordingto any one of claims 1-44, wherein the plant is wood or fiber crop. 53.The plant according to any one of claims 1-44, wherein the plant is anornamental plant.
 54. A part of the plant according to any one of claims1-53
 55. The plant according to claim 54 which is a pod, root, cutting,tuber, stem, stalk, fruit, berry, nut, flower, leaf, bark, wood,epidermis, vascular tissue, organ, protplast, crown, callus culture,petiole, petal, sepal, stamen, stigma, style, bud, meristem,cambium,pith, sheath, silk, or embryo.
 56. A meiocyte or gamete or ovuleor pollen or endosperm of the plant according to any one of claims 1-53.57. A seed, embryo or propagule of the plant according to anyone ofclaims 1-53.
 58. A progeny of the plant according to any one of claims1-53.
 59. The progeny of claim 58 wherein the progeny is the result ofself-breeding.
 60. The progeny of claim 58 wherein the progeny is theresult of cross-breeding.
 61. The progeny of claim 58 wherein theprogeny is the result of apomixis.
 62. The progeny of claim 58 whereinthe progeny is the result of clonal propagation.
 63. The progeny ofclaim 58 comprising a mini-chromosome descended from a parentalmini-chromosome that contained a centromere less than 150 kilobases inlength.
 64. The progeny of claim 58 comprising a mini-chromosomedescended from a parental mini-chromosome that contained a cetromereless than 100 kilobases in length.
 65. The progeny of claim 58comprising a mini-chromosome descended from a parental mini-chromosomethat contained a centromere less than 50 kilobases in length.
 66. Amethod of making a mini-chromosome for use in the plant according to anyone of claims 1-53 comprising identifying a centromere nucleotidesequence in a genomic DNA library using a multiplicity of diverseprobes, and constructing a mini-chromosome comprising the centromerenucleotide sequence.
 67. The method of claim 66 wherein the identifyingfurther comprises determining hybridization scores for hybridization ofthe multiplicity of diverse probes to genomic clones within the genomicDNA library, determining a classification for genomic clones within thegenomic DNA library according to the hybridization scores for at leasttwo of the diverse probes, and selecting one or more genomic cloneswithin one or more classifications for constructing the mini-chromosome.68. The method of claim 68 wherein at least three different probes areused.
 69. The method pf claim 68 wherein at least four different probesare used.
 70. The method of claim 69 wherein at least five differentprobes are used.
 71. The method of claim 70 wherein at least tendiffereent probes are used.
 72. The method of any one of the claims66-71 wherein at least one probe hybridizes to the centromere of achromosome.
 73. The method of any one of the claims 66-71 wherein atleast nr probe hybridizes to satellite repeat DNA.
 74. The method ofanyone of the claims 66-71 wherein at least one probe hybridizes toretroelement DNA.
 75. The method of any one of claims 66-71 wherein atleast one probe hybridizes to portions of genomic DNA that are heavilymethylated.
 76. The method of claim 66-71 wherein at least one probehybridizes to arrays of tandem repeats in genomic DNA.
 77. The method ofany one of claims 66-71 wherein at least one probe hybridizes toribosomal DNA, and a classification comprises a low hybridization scorefor hybridization to said probe.
 78. The method of any one claims 66-71wherein at least one probe hybridizes to mitochodrial DNA, and aclassification comprises a low hybridization score for hybridization tosaid probe.
 79. The method of any one of claims 66-71 wherein at leastone probe hybridizes to chloroplast DNA, and a classification comprisesa low hybridization score for hybridization to said probe.
 80. Themethod of any one of claims 66-71 wherein at least one probe hybridizesto telomere DNA.
 81. The method of any one of claims 66-71 wherein atleast one probe hybridizes to a pseudogene.
 82. A method of making aplant according to any one of claims 1-53 comprising delivering amini-chromosome to a plant cell using a biolistic method, wherein aparticle suitable for use in a biolistic method is delivered in a liquidwith the mini-chromosome, and
 83. The method of claim 82 wherein theliquid further comprises a divalent ion and a di- or poly-amine.
 84. Amethod of making a plant according to any one of claims 1-53 comprisingco-delivering to a plant cell a mini-chromosome and a nucleic acidencoding a growth inducing gene, wherein said nucleic acid is not partof the mini-chromosome, and regenerating a plant.
 85. The method ofclaim 84 wherein the nucleic acid encoding a growth inducing gene is notexpressed or not present in the regenerated plant.
 86. The method ofclaim 84 wherein the nucleic acid encoding a growth inducing gene isexpressed during regenerating the plant.
 87. The method of any one ofclaims 84-86 wherein the growth inducing gene is selected from the groupconsisting of encoding plant growth regulator genes,organogenesis-promoting, embryogenesis-promoting orregeneration-promoting genes
 88. The method of claim 87 wherein the geneis a Agrobacterium tumefaciens isopentenyl transferase gene,Agrobacterium rhizogenes isopentenyl transfersase gene, Agrobacteriumtumefaciens indole-3-acetamide hydrolase (IAAH) gene or Agrobacteriumtumefaciens tryptophan-2-monooxygenase (IAAM) gene.
 89. A method ofusing a plant according to any one of claims 1-53 to produce a foodproduct comprising the steps of growing the plant, and harvesting orprocessing the plant.
 90. A method of using a plant according to any oneof claims 1-53 to produce a recombinant protein comprising the step ofgrowing a plant comprising a mini-chromosome that comprises an exogenousnucleic acid encoding the recombinant protein.
 91. The method of claim90 further comprising the steps of harvesting the plant and isolatingthe recombinant protein from the plant.
 92. The method of claim 90 or 91wherein the recombinant protein is a pharmaceutical protein.
 93. Amethod of using a plant according to any one of claims 1-53 to produce achemical product comprising the step of growing a plant comprising amini-chromosome that comprises an exogenous nucleic acid encoding anenzyme involved in synthesis of the chemical product.
 94. The method ofclaim 93 further comprising the steps of harvesting the plant andisolating the chemical product form the plant.
 95. The method of claim93 or 94 wherein the chemical product is a pharmaceutical product.